Compositions and methods for enhancing drug delivery across and into ocular tissues

ABSTRACT

This invention provides compositions and methods for enhancing delivery of drugs and other agents across epithelial tissues, including into and across ocular tissues and the like. The compositions and methods are also useful for delivery across endothelial tissues, including the blood brain barrier. The compositions and methods employ a delivery enhancing transporter that has sufficient guanidino or amidino sidechain moieties to enhance delivery of a compound conjugated to the reagent across one or more layers of the tissue, compared to the non-conjugated compound. The delivery-enhancing polymers include, for example, poly-arginine molecules that are preferably between about 6 and 25 residues in length.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 09/792,480, filed on Feb. 23, 2001, which is acontinuation-in-part application of U.S. patent application Ser. No.09/648,400, filed on Aug. 24, 2000, which claims priority to U.S.Provisional Patent Application No. 60/150,510, filed Aug. 24, 1999. Bothof these applications are incorporated herein by reference for allpurposes

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to the field of compositions and methods thatenhance the delivery of drugs and other compounds across ocularepithelial and endothelial tissues as well as other tissues in the eyeand eye lid.

2. Background

Administration of drugs for treatment of the eye fall into at leastthree categories: topical administration, injection and systemicadministration.

Eye drops and ointments have been used over many years to treat a vastnumber of ocular disorders and diseases. However, topical administrationis not always effective because of the eye's natural protective surface.In many circumstances, less than one percent or less of the active agentis delivered to the target site.

Ocular injection can be effective to deliver a higher concentration ofthe active agent to the target. Sometimes it can be difficult toaccurately inject a drug to the correct site in the eye.

Systemic administration may be used to deliver agents to the eye, butrequires high doses because so little of the administrated compoundactually enters the eye.

The present invention addresses these and other problems.

SUMMARY OF THE INVENTION

The delivery-enhancing transporters and methods of the invention areuseful for delivering drugs, diagnostic agents, and other compounds ofinterest to the eye and other ocular tissues. In some embodiments, themethods involve administering to an ocular tissue a conjugate thatcomprises the compound and a delivery-enhancing transporter. Thedelivery-enhancing transporters, which are also provided by theinvention, have sufficient guanidino or amidino moieties to increasedelivery of the conjugate into the ocular tissue compared to delivery ofthe compound in the absence of the delivery-enhancing transporter. Insome embodiments, delivery of the conjugate into the ocular tissue isincreased at least two-fold compared to delivery of the compound in theabsence of the delivery-enhancing transporter. In some embodiments,delivery of the conjugate into the ocular tissue is increased at leastten-fold compared to delivery of the compound in the absence of thedelivery-enhancing transporter. In some embodiments, the ocular tissueis an ocular epithelial or endothelial tissue. In some embodiments, theocular tissue is the retina or the optic nerve.

The delivery-enhancing transporter and the compound are typicallyattached through a linker. In addition, the conjugate can comprise twoor more delivery-enhancing transporters linked to the compound.

Typically, the delivery-enhancing transporters comprise fewer than 50subunits and comprise at least 6 guanidino or amidino moieties. In someembodiments, the subunits are amino acids. In some embodiments, thedelivery-enhancing transporters have from 6 to 25 guanidino or amidinomoieties, and more preferably between 7 and 15 guanidino moieties andstill more preferably, at least six contiguous guanidino and/or amidinomoieties. In some embodiments, the delivery-enhancing transportersconsist essentially of 5 to 50 subunits, at least 50 of which compriseguanidino or amidino residues. In some of these embodiments, thesubunits are natural or non-natural amino acids. For example, in someembodiments, the delivery-enhancing transporter comprises 5 to 25arginine residues or analogs thereof. For example, the transporter cancomprise seven contiguous D-arginines.

In some embodiments, the delivery-enhancing transporter comprises 7-15arginine residues or analogs of arginine. The delivery-enhancingtransporter can have at least one arginine that is a D-arginine and insome embodiments, all arginines are D-arginine. In some embodiments, atleast 70% of the amino acids are arginines or arginine analogs. In someembodiments, the delivery-enhancing transporter comprises at least 5contiguous arginines or arginine analogs. The delivery-enhancingtransporters can comprise non-peptide backbones. In addition, in someaspects, the transporter is not attached to an amino acid sequence towhich the delivery-enhancing molecule is attached in a naturallyoccurring protein.

In some embodiments, the conjugate is administered as eye drops or as aninjection. The compounds of the conjugate include therapeutics for adisease selected from the group consisting of bacterial infections,viral infections, fungal infections, glaucoma, anterior, intermediate,and posterior uveitis, optic neuritis, Leber's neuroretinitis,retinitis, psudotumor/myositis, orbital myositis,hemangioma/lymphangioma, toxocariasis, Behcet's panuveitis, inflammatorychorisretinopathies, vasculitis, dry eye syndrome (Sjogren's syndrome),corneal edema, accommodative esotropia, cycloplegia, mydriasis, reversemydriasis, and macular degeneracy. In some embodiments, the compound isselected from the group consisting of anti-bacterial compounds,anti-viral compounds, anti-fungal compounds, anti-protozoan compounds,anti-histamines, compounds that dialate the pupil, anethsteticcompounds, steroidal antiinflammatory agents, antiinflammatoryanalgesics, chemotherapeutic agents, hormones, anticataract agents,neovascularization inhibitors, immunosuppressants, protease inhibitors,aldose reductase inhibitors, corticoid steroids, immunosuppressives,cholinergic agents, anticholinesterase agents, muscaric antagonists,sympathomimetic agents, α and β adrenergic antagonists, andanti-angiogenic factors. Thus, the compounds can include antibacterialcompounds, antiviral compounds, cyclosporin, ascomycins andcorticosteroids. In some embodiments, the compound is selected from thegroup consisting of acyclovir and cyclosporins.

As discussed above, the compound to be delivered can be connected to thedelivery-enhancing transporter by a linker. In some embodiments, thelinker is a releasable linker which releases the compound, inbiologically active form, from the delivery-enhancing transporter afterthe compound has passed into and/or through one or more layers of theepithelial and/or endothelial tissue. In some embodiments, the compoundis released from the linker by solvent-mediated cleavage. The conjugateis, in some embodiments, substantially stable at acidic pH but thecompound is substantially released from the delivery-enhancingtransporter at physiological pH. In some embodiments, the half-life ofthe conjugate is between 5 minutes and 24 hours upon contact with theskin or other epithelial or endothelial tissue. For example, thehalf-life can be between 30 minutes and 2 hours upon contact with theskin or other epithelial or endothelial tissue. In some embodiments, thelinker is stable in a saline solution a pH 7 but is cleaved whentransported into a cell.

Examples of conjugate structures of the invention include those havingstructures such as 3, 4, or 5, as follows:

wherein R¹ comprises the compound; X is a linkage formed between afunctional group on the biologically active compound and a terminalfunctional group on the linking moiety; Y is a linkage formed from afunctional group on the transport moiety and a functional group on thelinking moiety; A is N or CH; R² is hydrogen, alkyl, aryl, acyl, orallyl; R³ comprises the delivery-enhancing transporter; R⁴ is S, O, NR⁶or CR⁷R⁸; R⁵ is H, OH, SH or NHR⁶; R⁶ is hydrogen, alkyl, aryl, acyl orallyl; k and m are each independently selected from 1 and 2; and n is 1to 10.

Preferably, X is selected from the group consisting of —C(O)O—,—C(O)NH—, —OC(O)NH—, —S—S—, —C(S)O—, —C(S)NH—, —NHC(O)NH—, —SO₂NH—,—SONH—, phosphate, phosphonate phosphinate, and CR⁷R⁸, wherein R⁷ and R⁸are each independently selected from the group consisting of H andalkyl. In some embodiments, R₄ is S; R₅ is NHR₆; and R₆ is hydrogen,methyl, allyl, butyl or phenyl. In some embodiments, R₂ is benzyl; k, m,and n are each 1, and X is O. In some embodiments, the conjugatecomprises structure 3, Y is N, and R² is methyl, ethyl, propyl, butyl,allyl, benzyl or phenyl. In some embodiments, R² is benzyl; k, m, and nare each 1, and X is —OC(O)—. In some embodiments, the conjugatecomprises structure 4; R⁴ is S; R⁵ is NHR⁶; and R⁶ is hydrogen, methyl,allyl, butyl or phenyl. In some embodiments, the conjugate comprisesstructure 4; R⁵ is NHR⁶; R⁶ is hydrogen, methyl, allyl, butyl or phenyl;and k and m are each 1.

The invention also provides conjugates in which the release of thelinker from the biological agent involves a first, rate-limitingintramolecular reaction, followed by a faster intramolecular reactionthat results in release of the linker. The rate-limiting reaction can,by appropriate choice of substituents of the linker, be made to bestable at a pH that is higher or lower than physiological pH. However,once the conjugate has passed into and across one or more layers of anepithelial or endothelial tissue, the linker will be cleaved from theagent. An example of a compound that has this type of linker isstructure 6, as

wherein R′ comprises the compound; X is a linkage formed between afunctional group on the biologically active compound and a terminalfunctional group on the linking moiety; Y is a linkage formed from afunctional group on the transport moiety and a functional group on thelinking moiety; Ar is an aryl group having the attached radicalsarranged in an ortho or para configuration, which aryl group can besubstituted or unsubstituted; R³ comprises the delivery-enhancingtransporter; R⁴ is S, O, NR⁶ or CR⁷R⁸; R⁵ is H, OH, SH or NHR₆; R⁶ ishydrogen, alkyl, aryl, arylalkyl, acyl or allyl; R⁷ and R⁸ areindependently selected from hydrogen or alkyl; and k and m are eachindependently selected from 1 and 2.

In some embodiments, X is selected from the group consisting of —C(O)O—,—C(O)NH—, —OC(O)NH—, —S—S—, —C(S)O—, —C(S)NH—, —NHC(O)NH—, —SO₂NH—,—SONH—, phosphate, phosphonate phosphinate, and CR⁷R⁸, wherein R⁷ and R⁸are each independently selected from the group consisting of H andalkyl. In some embodiments, R⁴ is S; R⁵ is NHR⁶; and R⁶ is hydrogen,methyl, allyl, butyl or phenyl.

In preferred embodiments, the compositions of the invention comprise alinker susceptible to solvent-mediated cleavage. For example, apreferred linker is substantially stable at acidic pH but issubstantially cleaved at physiological pH. In some embodiments, thelinker is stable in a saline solution such as PBS. In some embodiments,the linker is stable in a saline solution but is cleaved whentransported into a cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a reaction scheme for the preparation of an α-chloroacetylcyclosporin A derivative.

FIG. 2 shows a general procedure for the coupling of cysteine-containingpeptides to the α-chloro acetyl cyclosporin A derivative.

FIG. 3 shows a reaction scheme for the coupling of the cyclosporin Aderivative to a biotin-labeled peptide.

FIG. 4 shows a reaction scheme for coupling of a cyclosporin Aderivative to an unlabeled peptide.

FIG. 5 A-H show various types of cleavable linkers that can be used tolink a delivery-enhancing transporter to a biologically active agent orother molecule of interest. FIG. 5A shows an example of a disulfidelinkage. FIG. 5B shows a photocleavable linker which is cleaved uponexposure to electromagnetic radiation. FIG. 5C shows a modified lysylresidue used as a cleavable linker. FIG. 5D shows a conjugate in whichthe delivery-enhancing transporter T is linked to the 2′-oxygen of theanticancer agent, paclitaxel. The linking moiety includes (i) a nitrogenatom attached to the delivery-enhancing transporter, (ii) a phosphatemonoester located para to the nitrogen atom, and (iii) a carboxymethylgroup meta to the nitrogen atom, which is joined to the 2′-oxygen ofpaclitaxel by a carboxylate ester linkage. FIG. 5E a linkage of adelivery-enhancing transporter to a biologically active agent, e.g.,paclitaxel, by an aminoalkyl carboxylic acid; a linker amino group isjoined to a delivery-enhancing transporter by an amide linkage and to apaclitaxel moiety by an ester linkage. FIGS. 5F and G show chemicalstructures and conventional numbering of constituent backbone atoms forpaclitaxel and “TAXOTERE™” (R′═H, R″═BOC). FIG. 5G shows the generalchemical structure and ring atom numbering for taxoid compounds.

FIG. 6 displays a synthetic scheme for a chemical conjugate between aheptamer of L-arginine and cyclosporin A (panel A) and its pH dependentchemical release (panel B). The α-chloro ester (6i) was treated withbenzylamine in the presence of sodium iodide to effect substitution,giving the secondary amine (6ii). Amine (6ii) was treated with anhydride(6) and the resultant crude acid (6iii) was converted to itscorresponding NHS ester (6iv). Ester (6iv) was then coupled with theamino terminus of hepta-L-arginine, giving the N-Boc protected CsAconjugate (6v). Finally, removal of the Boc protecting group with formicacid afforded the conjugate (6vi) as its octatrifluoroacetate salt afterHPLC purification.

FIG. 7 displays inhibition of inflammation in murine contact dermatitisby releasable R7 CsA. Balb/c (6-7 weeks) mice were painted on theabdomen with 100 μl of 0.7% DNFB in acetone olive oil (95:5). Three dayslater both ears of the animals were restimulated with 0.5% DNFB inacetone. Mice were treated one, five, and twenty hours afterrestimulation with either vehicle alone, 1% R7 peptide alone, 1% CsA, 1%nonreleasable R7 CsA, 0.01%/0.1%/1.0% releasable R7 CsA, and thefluorinated steroid positive control 0.1% triamcinolone acetonide. Earinflammation was measured 24 hours after restimulation using a springloaded caliper. The percent reduction of inflammation was calculatedusing the following formula (t−n)/(u−n), where t=thickness of thetreated ear, n=the thickness of a normal untreated ear, and u=thicknessof an inflamed ear without any treatment. N=20 animals in each group.

FIG. 8 shows a procedure for the preparation of acopper-diethylene-triaminepentaacetic acid complex (Cu-DTPA).

FIG. 9 shows a procedure for linking the Cu-DTPA to a transporterthrough an aminocaproic acid.

FIG. 10 shows a reaction for the acylation of hydrocortisone withchloroacetic anhydride.

FIG. 11 shows a reaction for linking the acylated hydrocortisone to atransporter.

FIG. 12 shows a reaction for preparation of C-2′ derivatives of taxol.

FIG. 13 shows a schematic of a reaction for coupling of a taxolderivative to a biotin-labeled peptide.

FIG. 14 shows a reaction for coupling of an unlabeled peptide to a C-2′derivative of taxol.

FIG. 15A-C shows a reaction scheme for the formation of other C-2′taxol-peptide conjugates.

FIG. 16 shows a general strategy for synthesis of a conjugate in which adrug or other biological agent is linked to a delivery-enhancingtransporter by a pH-releasable linker.

FIG. 17 shows a schematic diagram of a protocol for synthesizing a taxol2′-chloroacetyl derivative.

FIG. 18 shows a strategy by which a taxol 2′-chloroacetyl derivative islinked to an arginine heptamer delivery-enhancing transporter.

FIG. 19 shows three additional taxol-r7 conjugates that can be madeusing the reaction conditions illustrated in FIG. 18.

FIG. 20 shows the results of a 3 day MTT cytotoxicity assay using taxoland two different linkers.

FIG. 21 shows the FACS cellular uptake assay of truncated analogs ofTat₄₉₋₅₇ (Fl-ahx-RKKRRQRRR): Tat₄₉₋₅₆ (Fl-ahx-RKKRRQRR), Tat₄₉₋₅₅(Fl-ahx-RKKRRQR), Tat₅₀₋₅₇ (Fl-ahx-KKRRQRRR), and Tat₅₁₋₅₇(Fl-ahx-KRRQRRR). Jurkat cells were incubated with varyingconcentrations (12.5 μM shown) of peptides for 15 min at 23° C.

FIG. 22 shows FACS cellular uptake assay of alanine-substituted analogsof Tat₄₉₋₅₇: A-49 (Fl-ahx-AKKRRQRRR), A-50 (Fl-ahx-RAKRRQRRR), A-51(Fl-ahx-RKARRQRRR), A-52 (Fl-ahx-RKKARQRRR), A-53 (Fl-ahx-RKKRAQRRR),A-54 (Fl-ahx-RKKRRARRR), A-55 (Fl-ahx-RKKRRQARR), A-56(Fl-ahx-RKKRRQRAR), and A-57 (Fl-ahx-RKKRRQRRA). Jurkat cells wereincubated with varying concentrations (12.5 μM shown) of peptides for 12min at 23° C.

FIG. 23 shows the FACS cellular uptake assay of d- and retro-isomers ofTat₄₉₋₅₇: d-Tat49-57 (Fl-ahx-rkkrrqra), Tat57-49 (Fl-ahx-RRRQRRKKR), andd-Tat57-49 (Fl-ahx-rrrqrrkkr). Jurkat cells were incubated with varyingconcentrations (12.5 μM shown) of peptides for 15 min at 23° C.

FIG. 24 shows the FACS cellular uptake of a series of arginine oligomersand Tat₄₉₋₅₇: R5 (Fl-ahx-RRRRR), R6 (Fl-ahx-RRRRRR), R7(Fl-ahx-RRRRRRR), R8 (Fl-ahx-RRRRRRRR), R9 (Fl-ahx-RRRRRRRRR), r5(Fl-ahx-rrrrr), r6 r7 (Fl-r8 (Fl-ahx-rrrrrrrr), r9 (Fl-ahx-rrrrrrrrr).Jurkat cells were incubated with varying concentrations (12.5 μM shown)of peptides for 4 min at 23° C.

FIG. 25 displays the preparation of guanidine-substituted peptoids.

FIG. 26 displays the FACS cellular uptake of polyguanidine peptoids andd-arginine oligomers. Jurkat cells were incubated with varyingconcentrations (12.5 μM shown) of peptoids and peptides for 4 min at 23°C.

FIG. 27 displays the FACS cellular uptake of d-arginine oligomers andpolyguanidine peptoids. Jurkat cells were incubated with varyingconcentrations (12.5 μM shown) of fluorescently labeled peptoids andpeptides for 4 min at 23° C.

FIG. 28 displays the FACS cellular uptake of and d-arginine oligomersand N-hxg peptoids. Jurkat cells were incubated with varyingconcentrations (6.3 μM shown) of fluorescently labeled peptoids andpeptides for 4 min at 23° C.

FIG. 29 shows the FACS cellular uptake of d-arginine oligomers and N-chgpeptoids. Jurkat cells were incubated with varying concentrations (12.5μM shown) of fluorescently labeled peptoids and peptides for 4 min at23° C.

FIG. 30 shows a general strategy for attaching a delivery-enhancingtransporter to a drug that includes a triazole ring structure.

FIG. 31A and FIG. 31B show synthetic schemes for making conjugates inwhich FK506 is attached to a delivery-enhancing transporter.

FIG. 32 illustrates the conjugation of acyclovir to r7-amide via anN-terminal cysteine group. Conjugation with a biotin-containingtransporter is also shown.

FIG. 33 illustrates the conjugate formed between a retinal and a r9(shown without spacing amino acids).

FIG. 34 illustrates the use of a cleavable linker in preparing aretinoic acid-r9 conjugate.

FIG. 35 illustrates a method of linking active agents such as acyclovirto transport moieties.

FIG. 36 illustrates a method of linking active agents such as acyclovirto transport moieties.

FIG. 37 illustrates a method of linking active agents such as acorticoid steroid to transport moieties.

DETAILED DESCRIPTION Definitions

“Ocular tissue” refers to tissue of the eye and eyelid. Tissues orlayers of the eye include, e.g., the sclera, the cornea, which comprsiesa layer of nonkaratenized squamous epithelia, the corneal stroma,endotheliuim, including a cell layer lying on the thick basementmembrane (Descement's membrane). Additional ocular layers include, e.g.,The zona occludens, the aqueous humor, the oiris, the vitreoushummor/vitreous body, the choroid, the ciliary body including theciliary epithelium, the retina, including the rod and cone cells, thelens and the optic nerve. See. e.g., GRAY′S ANATOMY (Williams et al.,eds., 1995).

An “epithelial tissue” is the basic tissue that covers surface areas ofthe surface, spaces, and cavities of the body. Epithelial tissues arecomposed primarily of epithelial cells that are attached to one anotherand rest on an extracellular matrix (basement membrane) that istypically produced by the cells. Epithelial tissues include threegeneral types based on cell shape: squamous, cuboidal, and columnarepithelium. Squamous epithelium, which lines lungs and blood vessels, aswell as the cornea, is made up of flat cells. Cuboidal epithelium lineskidney tubules and is composed of cube shaped cells, while columnarepithelium cells line the digestive tract and have a columnarappearance. Epithelial tissues can also be classified based on thenumber of cell layers in the tissue. For example, a simple epithelialtissue is composed of a single layer of cells, each of which sits on thebasement membrane. A “stratified” epithelial tissue is composed ofseveral cells stacked upon one another; not all cells contact thebasement membrane. A “pseudostratified” epithelial tissue has cellsthat, although all contact the basement membrane, appear to bestratified because the nuclei are at various levels.

The term “trans-epithelial” delivery or administration refers to thedelivery or administration of agents by permeation through one or morelayers of a body surface or tissue, such as cornea, zona occludens, lensand the like, by topical administration. Delivery can be to a deeperlayer of the tissue, for example, and/or delivery to or from thebloodstream.

“Delivery enhancement, “penetration enhancement” or “permeationenhancement” as used herein relates to an increase in amount and/or rateof delivery of a compound that is delivered into and across one or morelayers of an epithelial or endothelial tissue or other ocular tissue. Anenhancement of delivery can be observed by measuring the rate and/oramount of the compound that passes through one or more layers of suchtissue. Delivery enhancement also can involve an increase in the depthinto the tissue to which the compound is delivered, and/or the extent ofdelivery to one or more cell types of the epithelial or other tissue(e.g., increased delivery to cornea, optic nerve, lens or other tissue).Such measurements are readily obtained by, for example, using adiffusion cell apparatus as described in U.S. Pat. No. 5,891,462.

The amount or rate of delivery of an agent across and/or into ocular orother epithelial or endothelial membrane is sometimes quantitated interms of the amount of compound passing through a predetermined area ofeye or other tissue. That area will usually be in the range of about 0.1cm² to about 100 cm², for example in the range of about 0.1 cm² to about1 cm², or in the range of about 0.5 cm² to about 2 cm².

The terms “guanidyl,” guanidinyl” and “guanidino” are usedinterchangeably to refer to a moiety having the formula —HN═C(NH₂)NH(unprotonated form). As an example, arginine contains a guanidyl(guanidino) moiety, and is also referred to as2-amino-5-guanidinovaleric acid or α-amino-6-guanidinovaleric acid.“Guanidium” refers to the positively charged conjugate acid form. Theterm “guanidino moiety” includes, for example, guanidine, guanidinium,guanidine derivatives such as (RNHC(NH)NHR′), monosubstitutedguanidines, monoguanides, biguanides, biguanide derivatives such as(RNHC(NH)NHC(NH)NHR'), and the like. In addition, the term “guanidinomoiety” encompasses any one or more of a guanide alone or a combinationof different guanides.

“Amidinyl” and “amidino” refer to a moiety having the formula—C(═NH)(NH₂). “Amidinium” refers to the positively charged conjugateacid form.

The term “trans-barrier concentration” or “trans-tissue concentration”refers to the concentration of a compound present on the side of one ormore layers of an epithelial or endothelial barrier tissue that isopposite or “trans” to the side of the tissue to which a particularcomposition has been added. For example, when a compound is applied tothe eye, the amount of the compound measured subsequently across one ormore layers of the eye is the trans-barrier concentration of thecompound.

“Biologically active agent” or “biologically active substance” refers toa chemical substance, such as a small molecule, macromolecule, or metalion, that causes an observable change in the structure, function, orcomposition of a cell upon uptake by the cell. Observable changesinclude increased or decreased expression of one or more mRNAs,increased or decreased expression of one or more proteins,phosphorylation of a protein or other cell component, inhibition oractivation of an enzyme, inhibition or activation of binding betweenmembers of a binding pair, an increased or decreased rate of synthesisof a metabolite, increased or decreased cell proliferation, and thelike.

The terms “therapeutic agent”, “therapeutic composition”, and“therapeutic substance” refer, without limitation, to any compositionthat can be used to the benefit of a mammalian species. Such agents maytake the form of ions, small organic molecules, peptides, proteins orpolypeptides, oligonucleotides, and oligosaccharides, for example.

The term “macromolecule” as used herein refers to large molecules (MWgreater than 1000 daltons) exemplified by, but not limited to, peptides,proteins, oligonucleotides and polynucleotides of biological orsynthetic origin.

“Small organic molecule” refers to a carbon-containing agent having amolecular weight (MW) of less than or equal to 1000 daltons.

The terms “non-polypeptide agent” and “non-polypeptide therapeuticagent” refer to the portion of a conjugate that does not include thedelivery-enhancing transporter, and that is a biologically active agentother than a polypeptide. An example of a non-polypeptide agent is ananti-sense oligonucleotide, which can be conjugated to a poly-argininepeptide to form a conjugate for enhanced delivery into and across one ormore layers of an epithelial or endothelial tissue.

A “subunit,” as used herein, is a monomeric unit that are joined to forma larger polymeric compound. The set of amino acids are an example ofsubunits. Each amino acid shares a common backbone (—C—C—N—), and thedifferent amino acids differ in their sidechains. The backbone isrepeated in a polypeptide. A subunit represents the shortest repeatingpattern of elements in a polymer backbone. For example, two amino acidsof a peptide are not considered a peptide because two amino acids wouldnot have the shortest repeating pattern of elements in the polymerbackbone.

The term “polymer” refers to a linear chain of two or more identical ornon-identical subunits joined by covalent bonds. A peptide is an exampleof a polymer; peptides can be composed of identical or non-identicalamino acid subunits that are joined by peptide linkages (amide bonds).

The term “peptide” as used herein refers to a compound made up of asingle chain of D- or L-amino acids or a mixture of D- and L-amino acidsjoined by peptide bonds. Generally, peptides contain at least two aminoacid residues and are less than about 50 amino acids in length. D-aminoacids are represented herein by a lower-case one-letter amino acidsymbol (e.g., r for D-arginine), whereas L-amino acids are representedby an upper case one-letter amino acid symbol (e.g., R for L-arginine).Homopolymer peptides are represented by a one-letter amino acid symbolfollowed by the number of consecutive occurrences of that amino acid inthe peptide- (e.g., R7 represents a heptamer that consists of L-arginineresidues).

The term “protein” as used herein refers to a compound that is composedof linearly arranged amino acids linked by peptide bonds, but incontrast to peptides, has a well-defined conformation. Proteins, asopposed to peptides, generally consist of chains of 50 or more aminoacids.

“Polypeptide” as used herein refers to a polymer of at least two aminoacid residues and which contains one or more peptide bonds.“Polypeptide” encompasses pep-tides and proteins, regardless of whetherthe polypeptide has a well-defined conformation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides compositions and methods that enhance thetransfer of compounds, including drugs and other biologically activecompounds, into and across one or more layers of an animal oculartissue, including epithelial or endothelial tissue, as well as otherocular tissue. The methods involve contacting the tissue with aconjugate that includes the compound of interest linked to adelivery-enhancing transporter. The delivery enhancing transportersprovided by the invention are molecules that include sufficientguanidino or amidino moieties to increase delivery of the conjugate intoand across one or more intact epithelial and endothelial tissue layers.The methods and compositions are useful for trans-epithelial andtrans-endothelial delivery of drugs and other biologically activemolecules, and also for delivery of imaging and diagnostic molecules.The methods and compositions of the invention are useful for delivery ofcompounds that require trans-epithelial or trans-endothelial transportto exhibit their biological effects, and that by themselves (withoutconjugation to a delivery-enhancing transporters or some othermodification), are unable, or only poorly able, to cross such tissuesand thus exhibit biological activity.

The delivery-enhancing transporters and methods of the invention providesignificant advantages over previously available methods for obtainingtrans-epithelial and trans-endothelial tissue delivery of compounds ofinterest. The transporters make possible the delivery of drugs and otheragents across tissues that were previously impenetrable to the drug. Forexample, while delivery of drugs across the retinal or cornealepithelium was previously nearly impossible for all but a few compounds,the methods of the invention can deliver compounds not only into cellsof a first layer of an epithelial tissue but also across one or morelayers of this layer.

The delivery-enhancing transporters increase delivery of the conjugateinto and across one or more intact epithelial or endothelial tissuelayers compared to delivery of the compound in the absence of thedelivery-enhancing transporter. The delivery-enhancing transporters can,in some embodiments, increase delivery of the conjugate significantlyover that obtained using the tat protein of HIV-1 (Frankel et al. (1991)PCT Pub. No. WO 91/09958). Delivery is also increased significantly overthe use of shorter fragments of the tat protein containing the tat basicregion (residues 49-57 having the sequence RKKRRQRRR) (Barsoum et al.(1994) WO 94/04686 and Fawell et al. (1994) Proc. Nat'l. Acad. Sci. USA91: 664-668). Preferably, delivery obtained using the transporters ofthe invention is increased more than 2-fold, still more preferablysix-fold, still more preferably ten-fold, and still more preferablytwenty-fold, over that obtained with tat residues 49-57. In someembodiments, the compositions of the invention do not include tatresidues 49-57.

Similarly, the delivery-enhancing transporters of the invention canprovide increased delivery compared to a 16 amino acidpeptide-cholesterol conjugate derived from the Antennapedia homeodomainthat is rapidly internalized by cultured neurons (Brugidou et al. (1995)Biochem. Biophys. Res. Commun. 214: 685-93). This region, residues 43-58at minimum, has the amino acid sequence RQIKIWFQNRRMKWKK. The Herpessimplex protein VP22, like tat and the Antennapedia domain, waspreviously known to enhance transport into cells, but was not known toenhance transport into and across endothelial and epithelial membranes(Elliot and O'Hare (1997) Cell 88: 223-33; Dilber et al. (1999) GeneTher. 6: 12-21; Phelan et al. (1998) Nature Biotechnol. 16: 440-3). Insome embodiments, the delivery-enhancing transporters providesignificantly increased delivery compared to the Antennapediahomeodomain and to the VP22 protein. In some embodiments, thecompositions of the invention do not include the Antennapediahomeodomain, the VP22 protein or eight contiguous arginines.

Structure of Delivery-Enhancing Transporters

The delivery-enhancing transporters of the invention are molecules thathave sufficient guanidino and/or amidino moieties to increase deliveryof a compound to which the delivery-enhancing transporter is attachedinto and across one or more layers of an ocular epithelial tissue or anendothelial tissue. The delivery-enhancing transporters generallyinclude a backbone structure to which is attached the guanidino and/oramidino sidechain moieties. In some embodiments, the backbone is apolymer that consists of subunits (e.g., repeating monomer units), atleast some of which subunits contain a guanidino or amidino moiety.

A. Guanidino and/or Amidino Moieties

The delivery-enhancing transporters typically display at least 5guanidino and/or amidino moieties, and more preferably 7 or more suchmoieties. Preferably, the delivery-enhancing transporters have 25 orfewer guanidino and/or amidino moieties, and often have 15 or fewer ofsuch moieties. In some embodiments, the delivery-enhancing transporterconsists essentially of 50 or fewer subunits, and can consistessentially of 25 or fewer, 20 or fewer, or 15 or fewer subunits. Thedelivery-enhancing transporter can be as short as 5 subunits, in whichcase all subunits include a guanidino or amidino sidechain moiety. Thedelivery-enhancing transporters can have, for example, at least 6subunits, and in some embodiments have at least 7 or 10 subunits.Generally, at least 50% of the subunits contain a guanidino or amidinosidechain moiety. More preferably, at least 70% of the subunits, andsometimes at least 90% of the subunits in the delivery-enhancingtransporter contain a guanidino or amidino sidechain moiety.

Some or all of the guanidino and/or amidino moieties in thedelivery-enhancing transporters can be contiguous. For example, thedelivery-enhancing transporters can include from 6 to 25 contiguousguanidino and/or amidino-containing subunits. Seven or more contiguousguanidino and/or amidino-containing subunits are present in someembodiments. In some embodiments, each subunit that contains a guanidinomoiety is contiguous, as exemplified by a polymer containing at leastsix contiguous arginine residues.

The delivery-enhancing transporters are exemplified by peptides.Arginine residues or analogs of arginine can constitute the subunitsthat have a guanidino moiety. Such an arginine-containing peptide can becomposed of either all D-, all L- or mixed D- and L-amino acids, and caninclude additional amino acids, amino acid analogs, or other moleculesbetween the arginine residues. Optionally, the delivery-enhancingtransporter can also include a non-arginine residue to which a compoundto be delivered is attached, either directly or through a linker. Theuse of at least one D-arginine in the delivery-enhancing transporterscan enhance biological stability of the transporter during transit ofthe conjugate to its biological target. In some cases thedelivery-enhancing transporters are at least about 50% D-arginineresidues, and for even greater stability transporters in which all ofthe subunits are D-arginine residues are used. If the delivery enhancingtransporter molecule is a peptide, the transporter is not attached to anamino acid sequence to which the amino acids that make up the deliveryenhancing transporter molecule are attached in a naturally occurringprotein.

Preferably, the delivery-enhancing transporter is linear. In a preferredembodiment, an agent to be delivered into and across one or more layersof an epithelial tissue is attached to a terminal end of thedelivery-enhancing transporter. In some embodiments, the agent is linkedto a single transport polymer to form a conjugate. In other embodiments,the conjugate can include more than one delivery-enhancing transporterlinked to an agent, or multiple agents linked to a singledelivery-enhancing transporter.

More generally, it is preferred that each subunit contains a highlybasic sidechain moiety which (i) has a pKa of greater than 11, morepreferably 12.5 or greater, and (ii) contains, in its protonated state,at least two geminal amino groups (NH₂) which share aresonance-stabilized positive charge, which gives the moiety a bidentatecharacter.

The guanidino or amidino moieties extend away from the backbone byvirtue of being linked to the backbone by a sidechain linker. Thesidechain atoms are preferably provided as methylene carbon atoms,although one or more other atoms such as oxygen, sulfur or nitrogen canalso be present. For example, a linker that attaches a guanidino moietyto a backbone can be shown as:

In these formulae, n is preferably at least 2, and is preferably between2 and 7. In some embodiments, n is 3 (arginine for structure 1). Inother embodiments, n is between 4 and 6; most preferably n is 5 or 6.Although the sidechain in the exemplified formulae is shown as beingattached to a peptide backbone (i.e., a repeating amide to which thesidechain is attached to the carbon atom that is α to the carbonylgroup, subunit 1) and a peptoid backbone (i.e., a repeating amide towhich the sidechain is attached to the nitrogen atom that is β to thecarbonyl group, subunit 2), other non-peptide backbones are alsosuitable, as discussed in more detail herein. Thus, similar sidechainlinkers can be attached to nonpeptide backbones (e.g., peptoidbackbones).

In some embodiments, the delivery-enhancing transporters are composed oflinked subunits, at least some of which include a guanidino and/oramidino moiety. Examples of suitable subunits having guanidino and/oramidino moieties are described below.

Amino Acids.

In some embodiments, the delivery-enhancing transporters are composed ofD or L amino acid residues. The amino acids can be naturally occurringor non-naturally occurring amino acids. Arginine(α-amino-δ-guanidinovaleric acid) and α-amino-ε-amidino-hexanoic acid(isosteric amidino analog) are examples of suitable guanidino- andamidino-containing amino acid subunits. The guanidinium group inarginine has a pKa of about 12.5. In some preferred embodiments thetransporters are comprised of at least six contiguous arginine residues.

Other amino acids, such as α-amino-β-guanidino-propionic acid,α-amino-γ-guanidino-butyric acid, or α-amino-ε-guanidino-caproic acid(containing 2, 3 or 5 sidechain linker atoms, respectively, between thebackbone chain and the central guanidinium carbon) can also be used.

D-amino acids can also be used in the delivery enhancing transporters.Compositions containing exclusively D-amino acids have the advantage ofdecreased enzymatic degradation. However, they can also remain largelyintact within the target cell. Such stability is generally notproblematic if the agent is biologically active when the polymer isstill attached. For agents that are inactive in conjugate form, a linkerthat is cleavable at the site of action (e.g., by enzyme- orsolvent-mediated cleavage within a cell) should be included within theconjugate to promote release of the agent in cells or organelles.

In addition, the transport moieties are amino acid oligomers of thefollowing formulae: (ZYZ)_(n)Z, (ZY)_(n)Z, (ZYY)_(n)Z and (ZYYY)_(n)Z.See, U.S. patent application Ser. No. 09/779,693, filed Feb. 7, 2001 andU.S. Patent Application No. 60/182,166, filed Feb. 14, 2000. “Z” in theformulae is D or L-arginine. “Y” is an amino acid that does not containa guanidyl or amidinyl moiety. The subscript “n” is an integer rangingfrom 2 to 25.

In the above transport moiety formulae, the letter “Y” represents anatural or non-natural amino acid. The amino acid can be essentially anycompound having (prior to incorporation into the transport moiety) anamino group (NH₂ or NH-alkyl) and a carboxylic acid group (CO₂H) and notcontaining either a guanidyl or amidinyl moiety. Examples of suchcompounds include D and L-alanine, D and L-cysteine, D and L-asparticacid, D and L-glutamic acid, D and L-phenylalanine, glycine, D andL-histidine, D and L-isoleucine, D and L-lysine, D and L-leucine, D andL-methionine, D and L-asparagine, D and L-proline, D and L-glutamine, Dand L-serine, D and L-threonine, D and L-valine, D and L-tryptophan, Dand L-hydroxyproline, D and L-tyrosine, sarcosine, β-alanine, γ-aminobutyric acid and ε-amino caproic acid. In each of the above formulae,each Y will be independent of any other Y present in the transportmoiety, though in some embodiments, all Y groups can be the same.

In one group of preferred embodiments, the transport moiety has theformula (ZYZ)_(n)Z, wherein each “Y” is independently selected fromglycine, β-alanine, γ-amino butyric acid and ε-amino caproic acid, “Z”is preferably L-arginine, and n is preferably an integer ranging from 2to 5. More preferably, each “Y” is glycine or ε-amino caproic acid and nis 3. Within this group of embodiments, the use of glycine is preferredfor those compositions in which the transport moiety is fused orcovalently attached directly to a polypeptide biological agent such thatthe entire composition can be prepared by recombinant methods. For thoseembodiments in which the transport moiety is to be assembled using, forexample, solid phase methods, ε-amino caproic acid is preferred.

In another group of preferred embodiments, the transport moiety has theformula (ZY)_(n)Z, wherein each “Y” is preferably selected from glycine,β-alanine, γ-amino butyric acid and ε-amino caproic acid, “Z” ispreferably L-arginine, and n is preferably an integer ranging from 4 to10. More preferably, each “Y” is glycine or ε-amino caproic acid and nis 6. As with the above group of specific embodiments, the use ofglycine is preferred for those compositions in which the transportmoiety is fused or covalently attached directly to a polypeptidebiological agent such that the entire composition can be prepared byrecombinant methods. For solution or solid phase construction of thetransport moiety, ε-amino caproic acid is preferred.

In yet another group of preferred embodiments, the transport moiety hasthe formula (ZYY)_(n)Z, wherein each “Y” is preferably selected fromglycine, β-alanine, γ-amino butyric acid and ε-amino caproic acid, “Z”is preferably L-arginine, and n is preferably an integer ranging from 4to 10. More preferably, each “Y” is glycine or ε-amino caproic acid andn is 6.

In still another group of preferred embodiments, the transport moietyhas the formula (ZYYY)_(n)Z, wherein each “Y” is preferably selectedfrom glycine, β-alanine, γ-amino butyric acid and ε-amino caproic acid,“Z” is preferably L-arginine, and n is preferably an integer rangingfrom 4 to 10. More preferably, “Y” is glycine and n is 6.

In other embodiments, each of the Y groups will be selected to enhancecertain desired properties of the transport moeity. For, example, whentransport moeities having a more hydrophobic character are desired, eachY can be selected from those naturally occurring amino acids that aretypically grouped together as hydrophobic amino acids (e.g.,phenylalanine; phenylglycine, valine, leucine, isoleucine). Similarly,transport moieties having a more hydrophilic character can be preparedwhen some or all of the Y groups are hydrophilic amino acids (e.g.,lysine, serine, threonine, glutamic acid, and the like).

One of skill in the art will appreciate that the transport moiety can bea polypeptide fragment within a larger polypeptide. For example, thetransport moiety can be of the formula (ZYY)_(n)Z yet have additionalamino acids which flank this moiety (e.g., X_(m)(ZYY)_(n)Z—X_(p) whereinthe subscripts m and p represent integers of zero to about 10 and each Xis independently a natural or non-natural amino acid).

Other Subunits.

Subunits other than amino acids can also be selected for use in formingtransport polymers. Such subunits can include, but are not limited to,hydroxy amino acids, N-methyl-amino acids amino aldehydes, and the like,which result in polymers with reduced peptide bonds. Other subunit typescan be used, depending on the nature of the selected backbone, asdiscussed in the next section.

B. Backbones

The guanidino and/or amidino moieties that are included in thedelivery-enhancing transporters are generally attached to a linearbackbone. The backbone can comprise a variety of atom types, includingcarbon, nitrogen, oxygen, sulfur and phosphorus, with the majority ofthe backbone chain atoms typically consisting of carbon. A plurality ofsidechain moieties that include a terminal guanidino or amidino groupare attached to the backbone. Although spacing between adjacentsidechain moieties is typically consistent, the delivery-enhancingtransporters used in the invention can also include variable spacingbetween sidechain moieties along the backbone.

A more detailed backbone list includes N-substituted amide (CONRreplaces CONH linkages), esters (CO₂), keto-methylene (COCH₂) reduced ormethyleneamino (CH₂NH), thioamide (CSNH), phosphinate (PO₂RCH₂),phosphonamidate and phosphonamidate ester (PO₂RNH), retropeptide (NHCO),trans-alkene (CR═CH), fluoroalkene (CF═CH), dimethylene (CH₂CH₂),thioether (CH₂S), hydroxyethylene (CH(OH)CH₂), methyleneoxy (CH₂O),tetrazole (CN₄), retrothioamide (NHCS), retroreduced (NHCH₂),sulfonamido (SO₂NH), methylenesulfonamido (CHRSO₂NH), retrosulfonamide(NHSO₂), and peptoids (N-substituted amides), and backbones withmalonate and/or gem-diamino-alkyl subunits, for example, as reviewed byFletcher et al. ((1998) Chem. Rev. 98:763) and detailed by referencescited therein. Many of the foregoing substitutions result inapproximately isosteric polymer backbones relative to backbones formedfrom α-amino acids.

As mentioned above, in a peptoid backbone, the sidechain is attached tothe backbone nitrogen atoms rather than the carbon atoms. (See e.g.,Kessler (1993) Angew. Chem. Int. Ed. Engl. 32:543; Zuckerman et al.(1992) Chemtracts-Macromol. Chem. 4:80; and Simon et al. (1992) Proc.Nat'l. Acad. Sci. USA 89:9367.) An example of a suitable peptoidbackbone is poly-(N-substituted)glycine (poly-NSG). Synthesis ofpeptoids is described in, for example, U.S. Pat. No. 5,877,278. As theterm is used herein, transporters that have a peptoid backbone areconsidered “non-peptide” transporters, because the transporters are notcomposed of amino acids having naturally occurring sidechain locations.Non-peptide backbones, including peptoid backbones, provide enhancedbiological stability (for example, resistance to enzymatic degradationin vivo).

C. Synthesis of Delivery-Enhancing Transporters

Delivery-enhancing transporters are constructed by any method known inthe art. Exemplary peptide polymers can be produced synthetically,preferably using a peptide synthesizer (e.g., an Applied BiosystemsModel 433) or can be synthesized recombinantly by methods well known inthe art. Recombinant synthesis is generally used when thedelivery-enhancing transporter is a peptide which is fused to apolypeptide or protein of interest.

N-methyl and hydroxy-amino acids can be substituted for conventionalamino acids in solid phase peptide synthesis. However, production ofdelivery-enhancing transporters with reduced peptide bonds requiressynthesis of the dimer of amino acids containing the reduced peptidebond. Such dimers are incorporated into polymers using standard solidphase synthesis procedures. Other synthesis procedures are well knownand can be found, for example, in Fletcher et al. (1998) Chem. Rev.98:763, Simon et al. (1992) Proc. Nat'l. Acad. Sci. USA 89:9367, andreferences cited therein.

The delivery-enhancing transporters of the invention can be flanked byone or more non-guanidino/non-amidino subunits (such as glycine,alanine, and cysteine, for example), or a linker (such as anaminocaproic acid group), that do not significantly affect the rate oftrans-tissue layer transport of the corresponding delivery-enhancingtransporter-containing conjugates. Also, any free amino terminal groupcan be capped with a blocking group, such as an acetyl or benzyl group,to prevent ubiquitination in vivo.

Where the transporter is a peptoid polymer, one synthetic methodinvolves the following steps: 1) a peptoid polyamine is treated with abase and pyrazole-1-carboxamidine to provide a mixture; 2) the mixtureis heated and then allowed to cool; 3) the cooled mixture is acidified;and 4) the acidified mixture is purified. Preferably the base used instep 1 is a carbonate, such as sodium carbonate, and heating step 2involves heating the mixture to approximately 50° C. for between about24 hours and about 48 hours. The purification step preferably involveschromatography (e.g., reverse-phase HPLC).

D. Attachment of Transport Polymers to Biologically Active Agents

The agent to be transported can be linked to the delivery-enhancingtransporter according to a number of embodiments. In one embodiment, theagent is linked to a single delivery-enhancing transporter, either vialinkage to a terminal end of the delivery-enhancing transporter or to aninternal subunit within the reagent via a suitable linking group.

In a second embodiment, the agent is attached to more than onedelivery-enhancing transporter, in the same manner as above. Thisembodiment is somewhat less preferred, since it can lead to crosslinkingof adjacent cells.

In a third embodiment, the conjugate contains two agent moietiesattached to each terminal end of the delivery-enhancing transporter. Forthis embodiment, it is presently preferred that the agent has amolecular weight of less than 10 kDa.

With regard to the first and third embodiments just mentioned, the agentis generally not attached to one any of the guanidino or amidinosidechains so that they are free to interact with the target membrane.

The conjugates of the invention can be prepared by straightforwardsynthetic schemes. Furthermore, the conjugate products are usuallysubstantially homogeneous in length and composition, so that theyprovide greater consistency and reproducibility in their effects thanheterogeneous mixtures.

According to an important aspect of the present invention, it has beenfound by the applicants that attachment of a single delivery-enhancingtransporter to any of a variety of types of biologically active agentsis sufficient to substantially enhance the rate of uptake of an agentinto and across one or more layers of epithelial and endothelialtissues, even without requiring the presence of a large hydrophobicmoiety in the conjugate. In fact, attaching a large hydrophobic moietycan significantly impede or prevent cross-layer transport due toadhesion of the hydrophobic moiety to the lipid bilayer of cells thatmake up the epithelial or endothelial tissue. Accordingly, the presentinvention includes conjugates that do not contain substantiallyhydrophobic moieties, such as lipid and fatty acid molecules.

Delivery-enhancing transporters of the invention can be attachedcovalently to biologically active agents by chemical or recombinantmethods.

1. Chemical Linkages

Biologically active agents such as small organic molecules andmacromolecules can be linked to delivery-enhancing transporters of theinvention via a number of methods known in the art (see, for example,Wong, S. S., Ed., Chemistry of Protein Conjugation and Cross-Linking,CRC Press, Inc., Boca Raton, Fla. (1991), either directly (e.g., with acarbodiimide) or via a linking moiety. In particular, carbamate, ester,thioether, disulfide, and hydrazone linkages are generally easy to formand suitable for most applications. Ester and disulfide linkages arepreferred if the linkage is to be readily degraded in the cytosol, aftertransport of the substance across the cell membrane.

Various functional groups (hydroxyl, amino, halogen, etc.) can be usedto attach the biologically active agent to the transport polymer. Groupsthat are not known to be part of an active site of the biologicallyactive agent are preferred, particularly if the polypeptide or anyportion thereof is to remain attached to the substance after delivery.

Polymers, such as peptides produced as described in PCT applicationUS98/10571 (Publication No. WO 9852614), are generally produced with anamino terminal protecting group, such as FMOC. For biologically activeagents which can survive the conditions used to cleave the polypeptidefrom the synthesis resin and deprotect the sidechains, the FMOC may becleaved from the N-terminus of the completed resin-bound polypeptide sothat the agent can be linked to the free N-terminal amine. In suchcases, the agent to be attached is typically activated by methods wellknown in the art to produce an active ester or active carbonate moietyeffective to form an amide or carbamate linkage, respectively, with thepolymer amino group. Of course, other linking chemistries can also beused.

To help minimize side-reactions, guanidino and amidino moieties can beblocked using conventional protecting groups, such as carbobenzyloxygroups (CBZ), di-t-BOC, PMC, Pbf, N—NO₂, and the like.

Coupling reactions are performed by known coupling methods in any of anarray of solvents, such as N,N-dimethyl formamide (DMF),N-methylpyrrolidinone, dichloromethane, water, and the like. Exemplarycoupling reagents include, for example, O-benzotriazolyloxytetramethyluronium hexafluorophosphate (HATU), dicyclohexylcarbodiimide, bromo-tris(pyrrolidino) phosphonium bromide (PyBroP), etc.Other reagents can be included, such as N,N-dimethylamino pyridine(DMAP), 4-pyrrolidino pyridine, N-hydroxy succinimide, N-hydroxybenzotriazole, and the like.

2. Fusion Polypeptides

Delivery-enhancing transporters of the invention can be attached tobiologically active polypeptide agents by recombinant means byconstructing vectors for fusion proteins comprising the polypeptide ofinterest and the delivery-enhancing transporter, according to methodswell known in the art. Generally, the delivery-enhancing transportercomponent will be attached at the C-terminus or N-terminus of thepolypeptide of interest, optionally via a short peptide linker.

3. Releasable Linkers

The biologically active agents are, in presently preferred embodiments,attached to the delivery-enhancing transporter using a linkage that isspecifically cleavable or releasable. The use of such linkages isparticularly important for biologically active agents that are inactiveuntil the attached delivery-enhancing transporter is released. In somecases, such conjugates that consist of a drug molecule that is attachedto a delivery-enhancing transporter can be referred to as prodrugs, inthat the release of the delivery-enhancing transporter from the drugresults in conversion of the drug from an inactive to an active form. Asused herein, “cleaved” or “cleavage” of a conjugate or linker refers torelease of a biological agent from a transporter molecule, therebyreleasing an active biological agent. “Specifically cleavable” or“specifically releasable” refers to the linkage between the transporterand the agent being cleaved, rather than the transporter being degraded(e.g., by proteolytic degradation).

In some embodiments, the linkage is a readily cleavable linkage, meaningthat it is susceptible to cleavage under conditions found in vivo. Thus,upon passing into and through one or more layers of an epithelial and/orendothelial tissue, the agent is released from the delivery-enhancingtransporter. Readily cleavable linkages can be, for example, linkagesthat are cleaved by an enzyme having a specific activity (e.g., anesterase, protease, phosphatase, peptidase, and the like) or byhydrolysis. For this purpose, linkers containing carboxylic acid estersand disulfide bonds are sometimes preferred, where the former groups arehydrolyzed enzymatically or chemically, and the latter are severed bydisulfide exchange, e.g., in the presence of glutathione. The linkagecan be selected so it is cleavable by an enzymatic activity that isknown to be present in one or more layers of an epithelial orendothelial tissue.

A specifically cleavable linker can be engineered onto a transportermolecule. For example, amino acids that constitute a proteaserecognition site, or other such specifically recognized enzymaticcleavage site, can be used to link the transporter to the agent.Alternatively, chemical or other types of linkers that are cleavable by,for example, exposure to light or other stimulus can be used to link thetransporter to the agent of interest.

A conjugate in which an agent to be delivered and a delivery-enhancingtransporter are linked by a specifically cleavable or specificallyreleasable linker will have a half-life. The term “half-life” in thiscontext refers to the amount of time required after applying theconjugate to an epithelial or endothelial membrane for one half of theamount of conjugate to become dissociated to release the free agent. Thehalf-life for some embodiments is between about 5 minutes and 24 hours,and, in some embodiments, is between 30 minutes and 2 hours. In someembodiments, the conjugate is stable in a buffered saline solution(e.g., PBS), but has a short half-life (e.g., less than one hour) in acell. The half-life of a conjugate can be “tuned” or modified, accordingto the invention, as described below.

In some embodiments, the cleavage rate of the linkers is pH dependent.For example, a linker can form a stable linkage between an agent and adelivery-enhancing transporter at an acidic pH (e.g., pH 6.5 or less,more preferably about 6 or less, and still more preferably about 5.5 orless). However, when the conjugate is placed at physiological pH (e.g.,pH 7 or greater, preferably about pH 7.4), the linker will undergocleavage to release the agent. Such pH sensitivity can be obtained by,for example, including a functional group that, when protonated (i.e.,at an acidic pH), does not act as a nucleophile.

At a higher (e.g., physiological) pH, the functional group is no longerprotonated and thus can act as a nucleophile. Examples of suitablefunctional groups include, for example, N and S. One can use suchfunctional groups to fine-tune the pH at which self-cleavage occurs.

In another embodiment, the linking moiety is cleaved throughself-immolation. Such linking moieties in a transportmoiety-biologically active compound conjugate contain a nucleophile(e.g., oxygen, nitrogen and sulfur) distal to the biologically activecompound and a cleavable group (e.g., ester, carbonate, carbamate andthiocarbamate) proximal to the biologically active compound.Intramolecular attack of the nucleophile on the cleavable group resultsin the scission of a covalent bond, thereby releasing the linking moietyfrom the biologically active compound.

Examples of conjugates containing self-immolating linking moieties(e.g., biologically active agent-L-transport moiety conjugates) arerepresented by structures 3, 4 and 5:

wherein: R′ is the biologically active compound; X is a linkage formedbetween a functional group on the biologically active compound and aterminal functional group on the linking moiety; Y is a linkage formedfrom a functional group on the transport moiety and a functional groupon the linking moiety; A is N or CH; R² is hydrogen, alkyl, aryl,arylalkyl, acyl or allyl; R³ is the transport moiety; R⁴ is S, O, NR⁶ orCR⁷R⁸; R⁵ is H, OH, SH or NHR⁶; R⁶ is hydrogen, alkyl, aryl, acyl orallyl; R⁷ and R⁸ are independently hydrogen or alkyl; k and m areindependently either 1 or 2; and n is an integer ranging from 1 to 10.Non-limiting examples of the X and Y linkages are (in eitherorientation): —C(O)O—, —C(O)NH—, —OC(O)NH—, —S—S—, —C(S)O—, —C(S)NH—,—NHC(O)NH—, —SO₂NH—, —SONH—, phosphate, phosphonate and phosphinate. Oneof skill in the art will appreciate that when the biological agent has ahydroxy functional group, then X will preferably be —OC(O)— or—OC(O)NH—. Similarly, when the linking group is attached to an aminoterminus of the transport moiety, Y will preferably be —C(O)NH—,—NHC(O)NH—, —SO₂NH—, —SONH— or —OC(O)NH— and the like. In each of thegroups provided above, NH is shown for brevity, but each of the linkages(X and Y) can contain substituted (e.g., N-alkyl or N-acyl) linkages aswell.

Turning first to linking groups illustrated by structure 3, an exampleand preferred embodiment is illustrated for formula 3a:

wherein the wavy lines indicate points of attachment to the transportmoiety and to the biologically active compound. Preparation of aconjugate containing this linking group is illustrated in Example 10(FIG. 6). In this Example and FIG. 6, cyclosporin A is treated withchloroacetic anhydride to form the chloroacetate ester 61 (numbering inFIG. 6) which is then combined with benzylamine to form the N-benzylglycine conjugate 6ii. Condensation of the glycine conjugate withBoc-protected diglycine anhydride provides the acid 6iii which isconverted to the more reactive N-hydroxy succinimide ester 61v and thencombined with the amino terminus of a transport moiety to form an amidelinkage. One of skill in the art will appreciate that the N-benzyl groupcan be replaced with other groups (e.g., alkyl, aryl, allyl and thelike) or that methylene groups can be replaced with, for example,ethylene, propylene and the like. Preferably, the methylene groups areretained as shown in 3a, to provide an appropriate steric or spatialorientation that allows the linkage to be cleaved in vivo (see FIG. 6B).

Accordingly, for structure 3, the following substituents are preferred:A is N; R² is benzyl; k, m and n are 1; X is —OC(O)— and Y is —C(O)NH—.

Linkages of structure 4, are exemplified by formula 4a:

wherein, as above, the wavy lines indicate the point of attachment toeach of the transport moiety and the biologically active agent. Thepreparation of conjugates having linking groups of formula 4a are shownin Examples 10-12. In Example 10 (see scheme in FIG. 32), acyclovir isacylated with α-loroacetic anhydride to form the achloroacetate ester32i. Reaction of 32i with a heptamer of D-arginine having an N-terminalcysteine residue, provides the thioether product 32ii. Alternatively,acyclovir can be attached to the C-terminus of a transport moiety usinga similar linkage formed between acyclovir α-hloroacetate ester and aheptamer of D-arginine having a C-terminal cysteine residue. In thisinstance, the cysteine residue is provided on the r₇ transport moiety asa C-terminal amide and the linkage has the form:

Accordingly, in one group of preferred embodiments, the conjugate isrepresented by formula 5, in which X is —OC(O)—; Y is —C(O)NH—; R⁴ is S;R⁵ is NHR⁶; and the subscripts k and m are each 1. In another group ofpreferred embodiments, the conjugate is represented by formula 2, inwhich X is —OC(O)—; Y is —NHC(O)—; R⁴ is S; R⁵ is CONH₂; and thesubscripts k and m are each 1. Particularly preferred conjugates arethose in which R⁶ is hydrogen, methyl, allyl, butyl or phenyl.

Linking groups represented by the conjugates shown in formula 6 aregenerally of the heterobifunctional type (e.g, ε-aminocaproic acid,serine, homoserine, γ-aminobutyric acid, and the like), althoughsuitably protected dicarboxylic acids or diamines are also useful withcertain biological agents.

For structure 6, the following substituents are preferred: R⁵ is NHR⁶,wherein R⁶ is hydrogen, methyl, allyl, butyl or phenyl; k is 2; X is—C(O)O—; and Y is —C(O)NH—.

Self-immolating linkers typically undergo intramolecular cleavage with ahalf-life between about 10 minutes and about 24 hours in water at 37° C.at a pH of approximately 7.4. Preferably, the cleavage half-life isbetween about 20 minutes and about 4 hours in water at 37° C. at a pH ofapproximately 7.4. More preferably, the cleavage half-life is betweenabout 30 minutes and about 2 hours in water at 37° C. at a pH ofapproximately 7.4.

For a conjugate having the structure 3, one can adjust the cleavagehalf-life by varying the R² substituent. By using an R² of increased ordecreased size, one can obtain a conjugate having a longer or shorterhalf-life respectively. R² in structure 3 is preferably methyl, ethyl,propyl, butyl, allyl, benzyl or phenyl.

Where there is a basic or acidic group in a self-immolating linker, onecan oftentimes adjust cleavage half-life according to the pH of theconjugate solution. For instance, the backbone amine group of structure3 is protonated at acidic pH (e.g., pH 5.5). The amine cannot serve as anucleophile inducing intramolecular cleavage when it is protonated. Uponintroduction of the conjugate into a medium at physiological pH (7.4),however, the amine is unprotonated a significant portion of the time.The cleavage half-life is correspondingly reduced.

In one embodiment, cleavage of a self-immolating linker occurs in twosteps: intramolecular reaction of a nucleophilic group resulting in thecleavage of a portion of the linking moiety; and, elimination of theremaining portion of the linking moiety. The first step of the cleavageis rate-limiting and can be fine-tuned for pH sensitivity and half-life.

Structure 6 is an example of a two-step, self-immolating moiety that isincorporated into a transport moiety-biologically active compoundconjugate:

wherein: R′ is the biologically active compound; X represents a linkagebetween a functional group on the biologically active compound and afunctional group on the linking moiety; Ar is a substituted orunsubstituted aryl group, wherein the methylene substituent and phenolicoxygen atom are either ortho or para to one another; R³ is the transportmoiety; R⁴ is S, O, NR⁶ or CR⁷R⁸; R⁵ is H, OH, SH or NHR⁶; R⁶ ishydrogen, alkyl, aryl, arylalkyl, acyl or allyl; R⁷ and R⁸ areindependently hydrogen or alkyl; and, k and m are independently either 1or 2.

An example of a suitable linking group to produce a conjugate of formula6 is:

The construction of a conjugate containing a linking group of formula 6ais provided in Example 14 (see also FIG. 34). In this example (andFigure), the α-chloroacetate ester of 2,4-dimethyl-4-hydroxymethylphenol(34i) is coupled to retinoic acid (34ii) using dicyclohexylcarbodiimide(DCC) and 4-dimethylaminopyridine (DMAP) to provide the intermediate34iii. Subsequent coupling of 34iii with a cysteine residue present onthe N-terminus of an arginine heptamer transport moiety provides thetarget conjugate 34iv.

Preferably, the linking groups used in the conjugates of formula 6, arethose in which Ar is an substituted or unsubstituted phenylene group; R⁴is S; R⁵ is NHR⁶, wherein R⁶ is hydrogen, methyl, allyl, butyl, acetylor phenyl; k and m are 1; X is —C(O)O—; and Y is —C(O)O— or —C(O)NH—.More preferably, R⁶ is hydrogen or acetyl.

While linking groups above have been described with reference toconjugates containing arginine heptamers, one of skill in the art willunderstand that the technology is readily adapted to conjugates with the“spaced” arginine transport moieties of the present invention.

Still other useful linking groups for use in the present invention havebeen described in copending PCT applications. See, for example PCTapplications US98/10571 (Publication No. WO 9852614) and US00/23440(Publication No. WO01/13957) which describe linking groups for similarcompositions, e.g., conjugates of biologically active agents andtransport oligomers. The linking technology described therein can beused in the present compositions in a similar manner.

Thus, in one group of embodiments, the linking moiety contains a firstcleavable group distal to the biologically active compound and a secondcleavable group proximal to the biologically active compound. Cleavageof the first cleavable group yields a nucleophile capable of reactingintramolecularly with the second cleavable group, thereby cleaving thelinking moiety from the biologically active compound. Examples ofmethods by which the first group is cleaved include photo-illuminationand enzyme mediated hydrolysis. This methodology has been illustratedfor various related small molecule conjugates discussed in PCTapplication US98/10571 (Publication No. WO 9852614).

In one approach, the conjugate can include a disulfide linkage, asillustrated in FIG. 5A of PCT application US00/23440 (Publication No.WO01/13957)), (see also, PCT application US98/10571 (Publication No. WO9852614)), which shows a conjugate (I) containing a transport polymer Twhich is linked to a cytotoxic agent, 6-mercaptopurine, by anN-acetyl-protected cysteine group which serves as a linker. Thus, thecytotoxic agent is attached by a disulfide bond to the 6-mercapto group,and the transport polymer is bound to the cysteine carbonyl moiety viaan amide linkage. Cleavage of the disulfide bond by reduction ordisulfide exchange results in release of the free cytotoxic agent. Amethod for synthesizing a disulfide-containing conjugate is provided inExample 9A of PCT application US98/10571. The product described thereincontains a heptamer of Arg residues which is linked to 6-mercaptopurineby an N-acetyl-Cys-Ala-Ala linker, where the Ala residues are includedas an additional spacer to render the disulfide more accessible tothiols and reducing agents for cleavage within a cell. The linker inthis example also illustrates the use of amide bonds, which can becleaved enzymatically within a cell.

In another approach, the conjugate includes a photocleavable linker thatis cleaved upon exposure to electromagnetic radiation. Application ofthis methodology is provided for a related system in FIG. 5B of PCTapplication US00/23440 (Publication No. WO01/13957) which shows aconjugate (II) containing a transport polymer T which is linked to6-mercaptopurine via a meta-nitrobenzoate linking moiety. Polymer T islinked to the nitrobenzoate moiety by an amide linkage to the benzoatecarbonyl group, and the cytotoxic agent is bound via its 6-mercaptogroup to the p-methylene group. The compound can be formed by reacting6-mercaptopurine with p-bromomethyl-m-nitrobenzoic acid in the presenceof NaOCH₃/methanol with heating, followed by coupling of the benzoatecarboxylic acid to a transport polymer, such as the amino group of aγ-aminobutyric acid linker attached to the polymer (see also, e.g.,Example 9B of PCT application US98/10571). Photo-illumination of theconjugate causes release of the 6-mercaptopurine by virtue of the nitrogroup that is ortho to the mercaptomethyl moiety. This approach findsutility in phototherapy methods as are known in the art, particularlyfor localizing drug activation to a selected area of the body.

In one group of preferred embodiments, the cleavable linker containsfirst and second cleavable groups that can cooperate to cleave theoligomer from the biologically active agent, as illustrated by thefollowing approaches. That is, the cleavable linker contains a firstcleavable group that is distal to the agent, and a second cleavablegroup that is proximal to the agent, such that cleavage of the firstcleavable group yields a linker-agent conjugate containing anucleophilic moiety capable of reacting intramolecularly to cleave thesecond cleavable group, thereby releasing the agent from the linker andoligomer.

Reference is again made to co-owned and copending PCT applicationUS00/23440 (Publication No. WO01/13957), in which FIG. 5C shows aconjugate (III) containing a transport polymer T linked to theanticancer agent, 5-fluorouracil (5FU). In that figure, the linkage isprovided by a modified lysyl residue. The transport polymer is linked tothe α-amino group, and the 5-fluorouracil is linked via the α-carbonyl.The lysyl ε-amino group has been modified to a carbamate ester ofo-hydroxymethyl nitrobenzene, which comprises a first, photolabilecleavable group in the conjugate. Photo-illumination severs thenitrobenzene moiety from the conjugate, leaving a carbamate that alsorapidly decomposes to give the free α-amino group, an effectivenucleophile. Intramolecular reaction of the α-amino group with the amidelinkage to the 5-fluorouracil group leads to cyclization with release ofthe 5-fluorouracil group.

Still other linkers useful in the present invention are provided in PCTapplication US00/23440 (Publication No. WO01/13957). In particular, FIG.5D of US00/23440 illustrates a conjugate (IV) containing adelivery-enhancing transporter T linked to 2′-oxygen of the anticanceragent, paclitaxel. The linkage is provided by a linking moiety thatincludes (i) a nitrogen atom attached to the delivery-enhancingtransporter, (ii) a phosphate monoester located para to the nitrogenatom, and (iii) a carboxymethyl group meta to the nitrogen atom, whichis joined to the 2′-oxygen of paclitaxel by a carboxylate ester linkage.Enzymatic cleavage of the phosphate group from the conjugate affords afree phenol hydroxyl group. This nucleophilic group then reactsintramolecularly with the carboxylate ester to release free paclitaxel,fully capable of binding to its biological target. Example 9C of PCTapplication US98/10571 describes a synthetic protocol for preparing thistype of conjugate.

Still other suitable linkers are illustrated in FIG. 5E of PCTapplication US00/23440 (Publication. No. WO01/13957). In the approachprovided therein, a delivery-enhancing transporter is linked to abiologically active agent, e.g., paclitaxel, by an aminoalkyl carboxylicacid. Preferably, the linker amino group is linked to the linkercarboxyl carbon by from 3 to 5 chain atoms (n=3 to 5), preferably either3 or 4 chain atoms, which are preferably provided as methylene carbons.As seen in FIG. 5E, the linker amino group is joined to thedelivery-enhancing transporter by an amide linkage, and is joined to thepaclitaxel moiety by an ester linkage. Enzymatic cleavage of the amidelinkage releases the delivery-enhancing transporter and produces a freenucleophilic amino group. The free amino group can then reactintramolecularly with the ester group to release the linker from thepaclitaxel.

In another approach, the conjugate includes a linker that is labile atone pH but is stable at another pH. For example, FIG. 6 of PCTapplication US00/23440 (Publication No. WO01/13957) illustrates a methodof synthesizing a conjugate with a linker that is cleaved atphysiological pH but is stable at acidic pH. Preferably, the linker iscleaved in water at a pH of from about 6.6 to about 7.6. Preferably thelinker is stable in water at a pH from about 4.5 to about 6.5.

Synthesis of other cleavable linkers and conjugates are described in,e.g., U.S. Pat. No. 6,306,993, issued Oct. 23, 2001.

Uses of Delivery-Enhancing Transporters

The delivery-enhancing transporters find use in therapeutic,prophylactic and diagnostic applications. The delivery-enhancingtransporters can carry a diagnostic or biologically active reagent intoand across one or more layers of epithelial tissue (e.g., ocular and thelike). This property makes the reagents useful for treating conditionsby delivering agents that must penetrate across one or more tissuelayers in order to exert their biological effect.

Moreover, the transporters of the present invention can also be usedalone, or in combination with another therapeutic or other compound, asa furin inhibitor. For example, in addition to various poly-argininetransporters, the synthetic transporters described herein, includingpeptoid and those transporters comprising non-naturally occurring aminoacids can be used to inhibit furins. See, e.g., Cameron et al., J. Biol.Chem. 275(47): 36741-9. Furins are proteases that convert a variety ofpro-proteins to their active components. Inhibition of furins is useful,for instance, for treating infections by viruses that rely on furinactivity for virulence or replication. See, e.g., Molloy, et al., T.Cell Biol. 9:28-35 (1999).

Similarly, the transporters of the invention are useful inhibitors ofcapthesin C. For example, certain poly arginine compounds are inhibitorsof capthesin C. See, e.g., Horn, et al., Eur. J. Biochem.267(11):3330-3336 (2000). Similarly, the transporters of the invention,including those comprising synthetic amino acids, are useful to inhibitcapthesin C.

Compositions and methods of the present invention have particularutility in the area of human and veterinary therapeutics. Generally,administered dosages will be effective to deliver picomolar tomicromolar concentrations of the therapeutic composition to the effectorsite. Appropriate dosages and concentrations will depend on factors suchas the therapeutic composition or drug, the site of intended delivery,and the route of administration, all of which can be derived empiricallyaccording to methods well known in the art. Further guidance can beobtained from studies using experimental animal models for evaluatingdosage, as are known in the art.

Administration of the compounds of the invention with a suitablepharmaceutical excipient as necessary can be carried out via any of theaccepted modes of administration. Thus, administration can be, forexample, topical, by intraocular injection, intravenous, subcutaneous,transcutaneous, intramuscular, oral, intra-joint, parenteral,peritoneal, intranasal, or by inhalation. Suitable sites ofadministration thus include, but are not limited to eye, skin orgastrointestinal tract (e.g., by mouth). The formulations may take theform of solid, semi-solid, lyophilized powder, or liquid dosage forms,such as, for example, tablets, pills, capsules, powders, solutions,suspensions, emulsions, creams, ointments, lotions, aerosols, eye drops,nasal spray or the like, preferably in unit dosage forms suitable forsimple administration of precise dosages. See, e.g., CLINICAL OPHTHALMICPHARMACOLOGY (Limberts & Potter, eds., 1987); PRINCIPLES OF INTERNALMEDICINE (Fauci et al., eds. 1998).

The compositions can include a conventional pharmaceutical carrier orexcipient and may additionally include other medicinal agents, carriers,adjuvants, and the like. Preferably, the composition will be about 0.1%to 75% by weight of a compound or compounds of the invention, with theremainder consisting of suitable pharmaceutical excipients. Appropriateexcipients can be tailored to the particular composition and route ofadministration by methods well known in the art, e.g., REMINGTON'SPHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co., Easton, Pa.(1990).

The aqueous suspensions of the present invention may contain, e.g.,compounds such as a buffer (e.g. carbonate salt, phosphate salt, acetatesalt, glutamic acid, citrate salt, ε-aminocaproic acid), an isotonizingagent (e.g., glycerol, mannitol, sorbitol, propylene glycol, sodiumchloride, potassium chloride, boric acid), a stabilizer (e.g., sodiumedetate, sodium citrate), a surfactant (e.g., polysorbate 80,polyoxyethylene(60) hydrogenated castor oil, tyloxapol, benzalkoniumchloride, polyoxyethylene fatty acid esters, polyoxyethylene alkylphenylethers, and polyoxyethylene alkyl ethers, mixtures thereof), apreservative (e.g., p-hydroxybenzoate and its analogs, benzalkoniumchloride, benzethonium chloride, chlorobutanol), a pH control agent(e.g., hydrochloric acid, sodium hydroxide, phosphoric acid), asurfactant polyoxyethylene fatty acid esters, and other additives.

Eye drops including, e.g., the conjugates of the invention, can alsoinclude an isotonic agent added to sterilized purified water, and ifrequired, a preservative, a buffering agent, a stabilizer, a viscousvehicle and the like are added to the solution and dissolved therein.After dissolution, the pH is adjusted with a pH controller to be withina range suitable for use as an ophthalmic medicine, preferably withinthe range of 4.5 to 8.

Sodium chloride, glycerin, mannitol or the like may be used as theisotonic agent; p-hydroxybenzoic acid ester, benzalkonium chloride orthe like as the preservative; sodium hydrogenphosphate, sodiumdihydrogenphosphate, boric acid or the like as the buffering agent;sodium edetate or the like as the stabilizer; polyvinyl alcohol,polyvinyl pyrrolidone, polyacrylic acid or the like as the viscousvehicle; and sodium hydroxide, hydrochloric acid or the like as the pHcontroller

For oral administration, such excipients include pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, andthe like. The composition may take the form of a solution, suspension,tablet, pill, capsule, powder, sustained-release formulation, and thelike.

In some embodiments, the pharmaceutical compositions take the form of apill, tablet or capsule, and thus, the composition can contain, alongwith the biologically active conjugate, any of the following: a diluentsuch as lactose, sucrose, dicalcium phosphate, and the like; adisintegrant such as starch or derivatives thereof; a lubricant such asmagnesium stearate and the like; and a binder such a starch, gum acacia,polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof.

The active compounds of the formulas may be formulated into asuppository comprising, for example, about 0.5% to about 50% of acompound of the invention, disposed in a polyethylene glycol (PEG)carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]).

Liquid compositions can be prepared by dissolving or dispersing compound(about 0.5% to about 20%), and optional pharmaceutical adjuvants in acarrier, such as, for example, aqueous saline (e.g., 0.9% w/v sodiumchloride), aqueous dextrose, glycerol, ethanol and the like, to form asolution or suspension, e.g., for intravenous administration.

If desired, the composition to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, such as, for example, sodium acetate,sorbitan monolaurate, or triethanolamine oleate.

For topical administration, the composition is administered in anysuitable format, such as a lotion or a transdermal patch. For deliveryby inhalation, the composition can be delivered as a dry powder (e.g.,Inhale Therapeutics) or in liquid form via a nebulizer.

Methods for preparing such dosage forms are known or will be apparent tothose skilled in the art; for example, see Remington's PharmaceuticalSciences, supra., and similar publications. The composition to beadministered will, in any event, contain a quantity of the pro-drugand/or active compound(s) in a pharmaceutically effective amount forrelief of the condition being treated when administered in accordancewith the teachings of this invention.

Generally, the compounds of the invention are administered in atherapeutically effective amount, i.e., a dosage sufficient to effecttreatment, which will vary depending on the individual and conditionbeing treated. Typically, a therapeutically effective daily dose is from0.1 to 100 mg/kg of body weight per day of drug. Most conditions respondto administration of a total dosage of between about 1 and about 30mg/kg of body weight per day, or between about 70 mg and 2100 mg per dayfor a 70 kg person.

Stability of the conjugate can be further controlled by the compositionand stereochemistry of the backbone and sidechains of thedelivery-enhancing transporters. For polypeptide delivery-enhancingtransporters, D-isomers are generally resistant to endogenous proteases,and therefore have longer half-lives in serum and within cells.D-polypeptide polymers are therefore appropriate when longer duration ofaction is desired. L-polypeptide polymers have shorter half-lives due totheir susceptibility to proteases, and are therefore chosen to impartshorter acting effects. This allows side-effects to be averted morereadily by withdrawing therapy as soon as side-effects are observed.Polypeptides comprising mixtures of D and L-residues have intermediatestabilities. Homo-D-polymers are generally preferred.

A. Ocular Administration

The delivery-enhancing transporters of the invention can be used toenhance administration of drugs through the tissues of the eye and otherrelated tissues such as the eye lid, as well as across the blood-brain,e.g., via the optic nerve. The ocular tissues include the cornea, iris,lens, vitreus, vitreus humor, the optic nerve and the eyelid.

Exemplary conjugates for administration to the eye include, e.g.,anti-bacterial compounds, anti-viral compounds, anti-fungal compounds,anti-protozoan compounds, anti-histamines, immunomodulatory compounds,compounds that dilate the pupil, anesthetic compounds, vitreous adductagents, steroidal antiinflammatory agents, antiinflammatory analgesics,chemotherapeutic agents, hormones, anticataract agents,neovascularization inhibitors, immunosuppressants, protease inhibitors,and aldose reductase inhibitors, corticoid steroids, immunosuppressives,cholinergic agents, anticholinesterase agents, muscaric antagonists,sympathomimetic agents, α and β adrenergic antagonists, andanti-angiogenic factors, among others.

Corticosteroids are useful for treating, e.g., inflammatory glaucoma,anterior, intermediate, and posterior uveitis, optic neuritis, Leber'sneuroretinitis, retinitis, pseudotumor/myositis, orbital myositis,hemangioma/lymphangioma, toxocariasisl Behcet's panuveitis, inflammatorychorisretinopathies, and vasculitis. Exemplary corticosteroids include,e.g., hydrocortisone, fludrocortisone, triamcinolone, dexamethasone,prednisolone, cortisone, aldosterone, and betamethasone.

Immunosuppressives are useful for treating, e.g., inflammatory glaucoma,anterior, intermediate, and posterior uveitis, optic neuritis, Leber'sneuroretinitis, retinitis, pseudotumor/myositis, orbital myositis,hemangioma/lymphangioma, toxocariasis, Behcet's panuveitis, inflammatorychorisretinopathies, vasculitis, and dry eye syndrome (Sjogren'ssyndrome). Dry eye, or “Sjogren's syndrome,” is an immune systemdisorder characterized by inflammation and dryness of the mouth, eyes,and other mucous membranes, damages the lacrimal glands, and this damageaffects tear production.

Exemplary immunosuppressives include, e.g., cyclosporins such ascyclosporin A, ascomycins such as FK-506, and nonsteroidalanti-inflammatory agents such as Cox-2 inhibitors, ketorolac, suprofen,and antazoline. Other exemplary immunosuppressives include, e.g.,rapamycin and tacrolimus.

Antibacterial agents are useful for treating, e.g., conjunctivitis,styes, blepharitis, and keratitis. Conjunctivitis, sometimes called pinkeye, is an inflammation of the blood vessels in the conjunctiva, themembrane that covers the sclera and inside of the eyelids.Conjunctivitis may be caused by bacteria or viruses.

Styes are noncontagious, bacterial infections of one of the sebaceousglands of the eyelid. A stye looks like a small, red bump either on theeyelid or on the edge of the eyelid.

Exemplary antibacterials include, e.g., beta-lactam antibiotics, such ascefoxitin, n-formamidoylthienamycin and other thienamycin derivatives,tetracyclines, chloramphenicol, neomycin, carbenicillin, colistin,penicillin G, polymyxin B, vancomycin, cefazolin, cephaloridine,chibrorifamycin, gramicidin, bacitracin, sulfonamides enoxacin,ofloxacin, cinoxacin, sparfloxacin, thiamphenicol, nalidixic acid,tosufloxacin tosilate, norfloxacin, pipemidic acid trihydrate, piromidicacid, fleroxacin, chlortetracycline, ciprofloxacin, erythromycin,gentamycin, norfloxacin, sulfacetamide, sulfixoxazole, tobramycin, andlevofloxacin.

Antiviral agents are useful for treating, e.g., Herpes simplexkeratitis, Herpes simplex conjunctivitis, Herpes zoster ophthalmicus andCytomegalovirus retinitis. Antiviral agents include, e.g., acyclovir,ganciclovir, didanosine, didovudine, idoxuridine, trifluridine,foscarnet, and vidarabine.

Antifungal agents are useful for treating, e.g., fungal keratitis andfungal endophthalmitis. Antifungal agents include, among others,polyenes such as amphotericin B and natamycin; imidazoles such asclotrimazole, miconazole, ketoconazole, fluconazole and econazole; andpyrimidines such as flucytosine. Other exemplary antifungal agentsincluded, e.g., itraconazole, flucytosine and pimaricin.

Antiparasitic compounds and/or anti-protozoal compounds include, e.g.,ivermectin, pyrimethamine, trisulfapidimidine, clindamycin andcorticosteroid preparations.

Cholinergic agents are useful for treating, e.g., glaucoma and cornealedema. Exemplary cholinergic agents include, e.g., acetylcholine,carbachol, and pilocarpine.

Anticholinesterase agents are useful for treating, e.g., glaucoma andaccommodative esotropia. Exemplary anticholinesterase agents include,e.g., physostigmine, demecarium, echothiophate, and isofluorophate.

Muscaric antagonists are useful for treating, e.g., cycloplegicretinoscopy and cycloplegia. They are also useful in dilated fundoscopicexams. Exemplary muscaric antagonists include, e.g., atropine,scopolamine, homatropine, cyclopentolate, and tropicamide.

Sympathomimetic agents are useful for treating, e.g., glaucoma andmydriasis. Exemplary sympathomimetic agents include, e.g., dipivefrin,epinephrine, phenylephrine, apraclonidine, cocaine, hydroxyamphetamine,naphazoline, and tetrahydrozoline.

α and β adrenergic antagonists are useful for treating, e.g., glaucomaand reverse mydriasis. Exemplary α and β adrenergic antagonists include,e.g., dapiprazole, betaxolol, carteolol, levobunolol, metipranolol, andtimolol.

Antiangiogenic factors are useful for treating, e.g., maculardegeneracy. Exemplary antiangiogenic factors include, e.g.,corticosteroids, thalidomide, and estradiols.

Antihistamines and decongestants include, e.g., pyrilamine,chlorpheniramine, tetrahydrazoline, antazoline and analogs thereof;mast-cell inhibitors of histamine release, such as cromolyn.

Antiinflammatory analgesics include, among others, alclofenac,aluminopropfen, ibuprofen, indomethacin, epirizole, oxaprozin,ketoprofen, diclofenac sodium, diflunisal, naproxen, piroxicam,fenbufen, flufenamic acid, flurbiprofen, floctafenine, pentazocine,metiazinic acid, mefenamic acid and mofezolac.

Other anesthetic agents include, e.g., cocaine, etidocaine cocaine,benoxinate, dibucaine hydrochloride, dyclonine hydrochloride, naepaine,phenacaine hydrochloride, piperocaine, proparacaine hydrochloride,tetracaine hydrochloride, hexylcaine, bupivacaine, lidocaine,mepivacaine and prilocaine.

Chemotherapeutic agents include, among others, sulfa drugs such assalazusulfapyridine, sulfadimethoxine, sulfamethizole, sulfamethoxazole,sulfamethopyrazine and sulfamonomethoxine.

Hormones include, among others, insulin zinc, testosterone propionateand estradiol benzoate.

Anticataract agents include, among others, pirenoxine and the like.

Neovascularization inhibitors include, among others, fumagillin andderivatives thereof.

Protease inhibitors include, among others,[L-3-trans-ethoxycarbonyloxiran-2-carbonyl]-L-leucine(3-methylbutyl)amide (E-64-d) and the like.

Aldose reductase inhibitors include, among others,5-(3-ethoxy-4-pentyloxyphenyl)thiazolidine-2,4-dione and the like.

Several classes of agents discussed above can be used to treat glaucoma.Glaucoma is a condition in which the normal fluid pressure inside theeyes (intraocular pressure, or IOP) slowly rises as a result of thefluid aqueous humor, which normally flows in and out of the eye, notbeing able to drain properly. Instead, the fluid collects and causespressure damage to the optic nerve and loss of vision. Useful compoundsto treat glaucoma, blindness, and other eye disorders include, e.g.,timolol, levobunolol and phenylepherine. Growth factors such as nervegrowth factor (NGF) (see, e.g., Bennett, et al. Mol Ther 1(6):501-5(2000)) are also useful for treating glaucoma and other oculardisorders. Antiglaucoma drugs, in addition to those discussed above,include, e.g., timalol, and its maleic salt and R-timolol and acombination of timolol or R-timolol with pilocarpine, as well as manyother adrenergic agonists and/or antagonists: epinephrine and anepinephrine complex, or prodrugs such as bitartrate, borate,hydrochloride and dipivefrine derivatives; carbonic anhydrase inhibitorssuch as acetazolamide, dichlorphenamide,2-(p-hydroxyphenyl)-thiothiophenesulfonamide,6-hydroxy-2-benzothiazolesulfonamide, and6-pivaloyloxy-2-benzothiazolesulfonamide.

Therapeutic compounds for treatment of ocular diseases, such as thosediscussed above, are well known to those of skill in the art. Typically,administration of the composition of the invention to the ocular tissuesis in the form of an eye drop. Alternatively, for example, thecompositions can be injected into the eye or applied as an ointment.

B. Diagnostic Imaging and Contrast Agents

The delivery-enhancing transporters of the invention are also useful fordelivery of diagnostic imaging and contrast agents into and across oneor more layers of ocular epithelial or endothelial tissue and within theeye and eye lid in general. Examples of diagnostic agents includesubstances that are labeled with radioactivity, such as ⁹⁹ mTcglucoheptonate, or substances used in magnetic resonance imaging (MRI)procedures such as gadolinium doped chelation agents (e.g. Gd-DTPA).Other examples of diagnostic agents include marker genes that encodeproteins that are readily detectable when expressed in a cell(including, but not limited to, (β-galactosidase, green fluorescentprotein, luciferase, and the like, as well as those compounds used toexamine the retina, such as sodium fluorescein; those used to examinethe conjunctiva, cornea and lacrimal apparatus, such as fluorescein androse bengal; and those used to examine abnormal pupillary responses suchas methacholine, cocaine, adrenaline, atropine, hydroxyamphetamine andpilocarpine. A wide variety of labels may be employed, such asradionuclides, fluors, enzymes, enzyme substrates, enzyme cofactors,enzyme inhibitors, ligands (particularly haptens), etc.

Biologically Active and Diagnostic Molecules Useful withDelivery-Enhancing Transporters

The delivery-enhancing transporters can be conjugated to a wide varietyof biologically active agents and molecules that have diagnostic use.

A. Small Organic Molecules

Small organic molecule therapeutic agents can be advantageously attachedto linear polymeric compositions as described herein, to facilitate orenhance transport across one or more layers of an epithelial orendothelial tissue. For example, delivery of highly charged agents, suchas levodopa (L-3,4-dihydroxy-phenylalanine; L-DOPA) may benefit bylinkage to delivery-enhancing transporters as described herein. Peptoidand peptidomimetic agents are also contemplated (e.g., Langston (1997)DDT 2:255; Giannis et al. (1997) Advances Drug Res. 29:1). Also, theinvention is advantageous for delivering small organic molecules thathave poor solubilities in aqueous liquids, such as serum and aqueoussaline. Thus, compounds whose therapeutic efficacies are limited bytheir low solubilities can be administered in greater dosages accordingto the present invention, and can be more efficacious on a molar basisin conjugate form, relative to the non-conjugate form, due to higheruptake levels by cells.

Since a significant portion of the topological surface of a smallmolecule is often involved, and therefore required, for biologicalactivity, the small molecule portion of the conjugate in particularcases may need to be severed from the attached delivery-enhancingtransporter and linker moiety (if any) for the small molecule agent toexert biological activity after crossing the target epithelial tissue.For such situations, the conjugate preferably includes a cleavablelinker for releasing free drug after passing through an epithelialtissue.

FIG. 5D and FIG. 5E are illustrative of another aspect of the invention,comprising taxane- and taxoid anticancer conjugates which have enhancedtrans-epithelial tissue transport rates relative to correspondingnon-conjugated forms. The conjugates are particularly useful forinhibiting growth of cancer cells. Taxanes and taxoids are believed tomanifest their anticancer effects by promoting polymerization ofmicrotubules (and inhibiting depolymerization) to an extent that isdeleterious to cell function, inhibiting cell replication and ultimatelyleading to cell death.

The term “taxane” refers to paclitaxel (FIG. 5F, R′=acetyl, R″=benzyl)also known under the trademark “TAXOL”) and naturally occurring,synthetic, or bioengineered analogs having a backbone core that containsthe A, B, C and D rings of paclitaxel, as illustrated in FIG. 5G. FIG.5F also indicates the structure of “TAXOTERE™” (R′═H, R″=BOC), which isa somewhat more soluble synthetic analog of paclitaxel sold byRhone-Poulenc. “Taxoid” refers to naturally occurring, synthetic orbioengineered analogs of paclitaxel that contain the basic A, B and Crings of paclitaxel, as shown in FIG. 5H. Substantial synthetic andbiological information is available on syntheses and activities of avariety of taxane and taxoid compounds, as reviewed in Suffness (1995)Taxol: Science and Applications, CRC Press, New York, N.Y., pp. 237-239,particularly in Chapters 12 to 14, as well as in the subsequentpaclitaxel literature. Furthermore, a host of cell lines are availablefor predicting anticancer activities of these compounds against certaincancer types, as described, for example, in Suffness at Chapters 8 and13.

The delivery-enhancing transporter is conjugated to the taxane or taxoidmoiety via any suitable site of attachment in the taxane or taxoid.Conveniently, the transport polymer is linked via a CT-oxygen atom,C7-oxygen atom, using linking strategies as above. Conjugation of atransport polymer via a C7-oxygen leads to taxane conjugates that haveanticancer and antitumor activity despite conjugation at that position.Accordingly, the linker can be cleavable or non-cleavable. Conjugationvia the C2′-oxygen significantly reduces anticancer activity, so that acleavable linker is preferred for conjugation to this site. Other sitesof attachment can also be used, such as C10.

It will be appreciated that the taxane and taxoid conjugates of theinvention have improved water solubility relative to taxol (0.25 μg/mL)and taxotere (6-7 μg/mL). Therefore, large amounts of solubilizingagents such as “CREMOPHOR EL” (polyoxyethylated castor oil), polysorbate80 (polyoxyethylene sorbitan monooleate, also known as “TWEEN 80”), andethanol are not required, so that side-effects typically associated withthese solubilizing agents, such as anaphylaxis, dyspnea, hypotension,and flushing, can be reduced.

B. Metals

Metals can be transported into and across one or more layers of ocularepithelia and endothelia using chelating agents such as texaphyrin ordiethylene triamine pentacetic acid (DTPA), conjugated to adelivery-enhancing transporter of the invention, as illustrated in theexamples. These conjugates are useful for delivering metal ions forimaging or therapy. Exemplary metal ions include Eu, Lu, Pr, Gd, Tc99m,Ga67, In111, Y90, Cu67, and Co57. Preliminary membrane-transport studieswith conjugate candidates can be performed using cell-based assays suchas described in the Example section below. For example, using europiumions, cellular uptake can be monitored by time-resolved fluorescencemeasurements. For metal ions that are cytotoxic, uptake can be monitoredby cytotoxicity.

C. Macromolecules

The enhanced transport methods of the invention are particularly suitedfor enhancing transport into and across one or more layers of anepithelial or endothelial tissue for a number of macromolecules,including, but not limited to proteins, nucleic acids, polysaccharides,and analogs thereof. Exemplary nucleic acids include oligonucleotidesand polynucleotides formed of DNA and RNA, and analogs thereof, whichhave selected sequences designed for hybridization to complementarytargets (e.g., antisense sequences for single- or double-strandedtargets), or for expressing nucleic acid transcripts or proteins encodedby the sequences. Analogs include charged and preferably unchargedbackbone analogs, such as phosphonates (preferably methyl phosphonates),phosphoramidates (N3′ or N5′), thiophosphates, unchargedmorpholino-based polymers, and protein nucleic acids (PNAs). Suchmolecules can be used in a variety of therapeutic regimens, includingenzyme replacement therapy, gene therapy, and anti-sense therapy, forexample.

By way of example, protein nucleic acids (PNA) are analogs of DNA inwhich the backbone is structurally homomorphous with a deoxyribosebackbone. The backbone consists of N-(2-aminoethyl)glycine units towhich the nucleobases are attached. PNAs containing all four naturalnucleobases hybridize to complementary oligonucleotides obeyingWatson-Crick base-pairing rules, and is a true DNA mimic in terms ofbase pair recognition (Egholm et al. (1993) Nature 365:566-568). Thebackbone of a PNA is formed by peptide bonds rather than phosphateesters, making it well-suited for anti-sense applications. Since thebackbone is uncharged, PNA/DNA or PNA/RNA duplexes that form exhibitgreater than normal thermal stability. PNAs have the additionaladvantage that they are not recognized by nucleases or proteases. Inaddition, PNAs can be synthesized on an automated peptides synthesizerusing standard t-Boc chemistry. The PNA is then readily linked to atransport polymer of the invention.

Examples of anti-sense oligonucleotides whose transport into and acrossepithelial and endothelial tissues can be enhanced using the methods ofthe invention are described, for example, in U.S. Pat. No. 5,594,122.Such oligonucleotides are targeted to treat human immunodeficiency virus(HIV). Conjugation of a transport polymer to an anti-senseoligonucleotide can be effected, for example, by forming an amidelinkage between the peptide and the 5′-terminus of the oligonucleotidethrough a succinate linker, according to well-established methods. Theuse of PNA conjugates is further illustrated in Example 11 of PCTApplication PCT/US98/10571. FIG. 7 of that application shows resultsobtained with a conjugate of the invention containing a PNA sequence forinhibiting secretion of gamma-interferon (γ-IFN) by T cells, as detailedin Example 11. As can be seen, the anti-sense PNA conjugate waseffective to block γ-IFN secretion when the conjugate was present atlevels above about 10 μM. In contrast, no inhibition was seen with thesense-PNA conjugate or the non-conjugated antisense PNA alone.

Another class of macromolecules that can be transported across one ormore layers of an epithelial or endothelial tissue is exemplified byproteins, and in particular, enzymes. Therapeutic proteins include, butare not limited to replacement enzymes. Therapeutic enzymes include, butare not limited to, alglucerase, for use in treating lysozomalglucocerebrosidase deficiency (Gaucher's disease), alpha-L-iduronidase,for use in treating mucopolysaccharidosis I,alpha-N-acetylglucosamidase, for use in treating sanfilippo B syndrome,lipase, for use in treating pancreatic insufficiency, adenosinedeaminase, for use in treating severe combined immunodeficiencysyndrome, and triose phosphate isomerase, for use in treatingneuromuscular dysfunction associated with triose phosphate isomerasedeficiency.

In addition, and according to an important aspect of the invention,protein antigens may be delivered to the cytosolic compartment ofantigen-presenting cells (APCs), where they are degraded into peptides.The peptides are then transported into the endoplasmic reticulum, wherethey associate with nascent HLA class 1 molecules and are displayed onthe cell surface. Such “activated” APCs can serve as inducers of class Irestricted antigen-specific cytotoxic T-lymphocytes (CTLs), which thenproceed to recognize and destroy cells displaying the particularantigen. APCs that are able to carry out this process include, but arenot limited to, certain macrophages, B cells and dendritic cells. In oneembodiment, the protein antigen is a tumor antigen for eliciting orpromoting an immune response against tumor cells. The transport ofisolated or soluble proteins into the cytosol of APC with subsequentactivation of CTL is exceptional, since, with few exceptions, injectionof isolated or soluble proteins does not result either in activation ofAPC or induction of CTLs. Thus, antigens that are conjugated to thetransport enhancing compositions of the present invention may serve tostimulate a cellular immune response in vitro or in vivo.

In another embodiment, the invention is useful for deliveringimmunospecific antibodies or antibody fragments to the cytosol tointerfere with deleterious biological processes such as microbialinfection. Recent experiments have shown that intracellular antibodiescan be effective antiviral agents in plant and mammalian cells (e.g.,Tavladoraki et al. (1993) Nature 366:469; and Shaheen et al. (1996) J.Virol. 70:3392. These methods have typically used single-chain variableregion fragments (scFv), in which the antibody heavy and light chainsare synthesized as a single polypeptide. The variable heavy and lightchains are usually separated by a flexible linker peptide (e.g., of 15amino acids) to yield a 28 kDa molecule that retains the high affinityligand binding site. The principal obstacle to wide application of thistechnology has been efficiency of uptake into infected cells. But byattaching transport polymers to scFv fragments, the degree of cellularuptake can be increased, allowing the immunospecific fragments to bindand disable important microbial components, such as HIV Rev, HIV reversetranscriptase, and integrase proteins.

D. Peptides

Peptides to be delivered by the enhanced transport methods describedherein include, but should not be limited to, effector polypeptides,receptor fragments, and the like. Examples include peptides havingphosphorylation sites used by proteins mediating intra-cellular signals.Examples of such proteins include, but are not limited to, proteinkinase C, RAF-1, p21Ras, NF-κB, C-JUN, and cytoplasmic tails of membranereceptors such as IL-4 receptor, CD28, CTLA-4, V7, and MHC Class I andClass II antigens.

When the delivery-enhancing transporter is also a peptide, synthesis canbe achieved either using an automated peptide synthesizer or byrecombinant methods in which a polynucleotide encoding a fusion peptideis produced, as mentioned above.

EXAMPLES

The following examples are offered to illustrate, but not to limit thepresent invention.

Example 1 Ocular Delivery of Transporter Conjugates

The ability of the transporters of the invention to penetrate thetissues of the eye was examined. Biotinylated r8 was both injected intothe eyes of rabbits and also applied as eyedrops to the outside of theeye.

Briefly, 5 drops of a 1 mM solution of biotinylated r8 in PBS wasapplied to both eyes of a rabbit and allowed to incubate 15 minutes. Theanimal was sacrificed and one eye was dissected intact with adjacenttissue, whereas the other was separated into each of its componentparts, frozen and separately sectioned, stained withstreptavidin-fluorescein and counterstained with propidium iodide.Results demonstrated staining in the cornea and eyelid, but not thelens.

Fifty microliters of a 10 mM solution of biotinylated r8 in PBS wasinjected into the vitreus humor of another animal and the animal wassacrificed 30 minutes later and the injected eye was dissected. Againone orbital was frozen intact while the other was dissected and thecomponents separately frozen. Results from the injection experimentsdemonstrated that all interior surfaces of the orb were stained.

Example 2 Synthesis, In Vitro and In Vivo Activity of a ReleasableConjugate of a Short Oligomer of Arginine and CsA

Modification of the 2° alcohol of Cyclosporin A results in significantloss of its biological activity. See, e.g., R. E. Handschumacher, etal., Science 226, 544-7 (1984). Consequently, to ensure release of freeCyclosporin A from its conjugate after transport into cells, CyclosporinA was conjugated to an oligo-arginine transporter through a pH sensitivelinker as shown in FIG. 10. The resultant conjugate is stable at acidicpH but at pH>7 it undergoes an intramolecular cyclization involvingaddition of the free amine to the carbonyl adjacent to Cyclosporin A(FIG. 6), which results in the release of unmodified Cyclosporin A.

Another modification in the design of the releasable conjugate was theuse of L-arginine (R), and not D-arginine (r) in the transporter. Whilethe oligo-D-arginine transporters were used for the histologicalexperiments to ensure maximal stability of the conjugate and thereforeaccuracy in determining its location through fluorescence, oligomers ofL-arginine were incorporated into the design of the releasable conjugateto minimize its biological half-life. Consistent with its design, theresultant releasable conjugate was shown to be stable at acidic pH, butlabile at physiological pH in the absence of serum. This releasableCyclosporin A conjugate's half-life in pH 7.4 PBS was 90 minutes.

Results

The releasable guanidino-heptamer conjugate of Cyclosporin A was shownto be biologically active by inhibiting IL-2 secretion by the human Tcell line, Jurkat, stimulated with PMA and ionomycin in vitro. See R.Wiskocil, et al., J Immunol 134, 1599-603 (1985). The conjugate wasadded 12 hours prior to the addition of PMA/ionomycin and dose dependentinhibition was observed by the releasable R7 CsA conjugate. Thisinhibition was not observed with a nonreleasable analog (FIG. 6) thatdiffered from the releasable conjugate by retention of the t-Bocprotecting group, which prevented cyclization and resultant release ofthe active drug. The EC₅₀ of the releasable R7 cyclosporin conjugate wasapproximately two fold higher than CsA dissolved in alcohol and added atthe same time as the releasable conjugate.

The releasable R7 CsA conjugate was assayed in vivo for functionalactivity using a murine model of contact dermatitis. Treatment with the1% releasable R7CSA conjugate resulted in 73.9%±4.0 reduction in earinflammation (FIG. 7). No reduction in inflammation was seen in theuntreated ear, indicating that the effect seen in the treated ear waslocal and not systemic. Less inhibition was observed in the ears of micetreated with 0.1 and 0.01% R7-CsA (64.8%±4.0 and 40.9%±3.3respectively), demonstrating that the effect was titratable. Treatmentwith the fluorinated corticosteroid positive control resulted inreduction in ear swelling (34.1%±6.3), but significantly less than thatobserved for 0.1% releasable R7 CsA (FIG. 7). No reduction ofinflammation was observed in any of the mice treated with unmodifiedCyclosporin A, vehicle alone, R7, or nonreleasable R7 CsA.

Example 3 The Preparation of Copper and Gadolinium-DTPA-r7 ComplexesMethods

1. Preparation of Metal Complexes

Step 1—Preparation of Copper-Diethylenetriaminepentaacetic Acid Complex(Cu-DTPA)

Copper carbonate (10 mmol) and diethylenetriaminpentacetic acid (10mmol) were dissolved in water (150 mL) (FIG. 8). After 18 h, thesolution was centrifuged to removed any solids. The blue solution wasdecanted and lyophilized to provide a blue powder (yields>90%).

Step 2—Preparation of DTPA Transporter

The Cu-DTPA was linked to a transporter through an aminocaproic acidspacer using a PE Applied Biosystems Peptide Synthesizer (ABI 433A)(FIG. 9). The material was cleaved from the resin by treatment withtrifluoroacetic acid (TFA) (40 mL), triisopropyl silane (1004) andphenol (100 μL) for 18 h. The resin was filtered off and the peptide wasprecipitated by addition of diethyl ether (80 mL). The solution wascentrifuged and the solvent decanted off. The crude solid was purifiedby reverse-phase HPLC using a water/acetonitrile gradient. Treatmentwith TFA resulted in loss of Cu²⁺ ion which needed to be reinserted.

DTPA-aca-R7-CO2H (10 mg, 0.0063 mmol) and copper sulfate (1.6 mg, 0.0063mmol) were dissolved in water (1 mL). Let gently stir for 18 h andlyophilized to provide product as a white powder (10 mg).

2. Analysis of Transport Across Skin

Metal diethylenetriaminepentaacetic acid (DTPA) complexes were formed bymixing equimolar amounts of metal salts with DTPA in water for 18 hours.At the end of this time, the solutions were centrifuged, frozen andlyophilized. The dried powder was characterized by mass spectrometry andused in solid phase peptide synthesis. The metal-DTPA complexes wereattached to polymers of D- or L-arginine that were still attached tosolid-phase resin used in peptide synthesis. The metal-DTPA complexeswere attached using an aminocaproic acid spacer.

Peptides were synthesized using solid phase techniques and commerciallyavailable Fmoc amino acids, resins, and reagents (PE Biosystems, FosterCity Calif., and Bachem Torrence, Calif.) on a Applied Biosystems 433peptide synthesizer. Fastmoc cycles were used withO-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexyluorophosphate(HATU) substituted for HBTU/HOBt as the coupling reagent. Prior to theaddition of biotin to the amino terminus of the peptide, amino caproicacid (aca) was conjugated and acted as a spacer. The peptides werecleaved from the resin using 96% trifluoroacetic acid, 2% triisopropylsilane, and 2% phenol for between 1 and 12 hours, also releasing themetal. The longer reaction times were necessary to completely remove thePbf protecting groups from the polymers of arginine. The peptidessubsequently were filtered from the resin, precipitated using diethylether, purified using HPLC reverse phase columns (Alltech Altima,Chicago, Ill.) and characterized using either electrospray or matrixassisted laser desorption mass spectrometry (Perceptive Biosystems,Boston, Mass.).

The metal is replaced after HPLC purification and lyophilization of thepeptide-DTPA complex. Replacement of the metal involved incubation ofequimolar amounts of the metal salt with the peptide-aminocaproic acid-DTPA complex and subsequent lyophilization.

Example 4 Conjugate of Taxol and Delivery-Enhancing Transporter withpH-Releasable Linker

This Example demonstrates the use of a general strategy for synthesizingprodrugs that have a delivery-enhancing transporter linked to a drug bya linker that releases the drug from the delivery-enhancing transporterupon exposure to physiological pH. In general, a suitable site on thedrug is derivatized to carry an α-chloroacetyl residue. Next, thechlorine is displaced with the thiol of a cysteine residue that carriesan unprotected amine. This scheme is shown in FIG. 16.

Methods Synthesis of Taxol-2′-chloroacetyl

Taxol (89.5 mg, 104.9 μmol) was dissolved in CH₂Cl₂ (3.5 mL). Thesolution was cooled to 0° C. under an N₂-atmosphere. α-Chloroaceticanhydride (19.7 mg, 115.4 μmol) was added, followed by DIEA (14.8 mg,115.4 μmol). The solution was allowed to warm to room temperature. Afterthin layer chromatography (tlc) analysis indicated complete consumptionof starting material, the solvent was removed in vacuo and the crudematerial was purified by flash chromatography on silica gel (eluent:EtOAC/Hex 20%-50%) yielding the desired material (99.8 mg, quantitative)(FIG. 18).

¹H-NMR (CDCl₃): δ=8.13 (d, J=7.57 Hz, 2H), 7.72 (d, J=7.57 Hz, 2H),7.62-7.40 (m, 11H), 6.93 (d, J=9.14 Hz, 1H), 6.29-6.23 (m, 2H), 6.01 (d,J=7.14 Hz, 1H), 5.66 (d, J=6.80 Hz, 1H), 5.55 (d, J=2.24 Hz, 1H), 4.96(d, J=8.79 Hz, 1H), 4.43 (m, 1H), 4.30 (d, J=8.29 Hz, 1H), 4.20-4.15 (m,2H), 3.81 (d, J=6.71 Hz, 1H), 2.56-2.34 (m, 3H), 2.45 (s, 3H), 2.21 (s,3H), 2.19 (m, 1H), 1.95-1.82 (m, 3H), 1.92 s, (3H), 1.67 (s, 3H), 1.22(s, 3H), 1.13 (s, 3H) ppm.

¹³C-NMR (CDCl₃): δ=203.6, 171.1, 169.7, 167.3, 167.0, 166.9, 166.3,142.3, 136.4, 133.6, 133.5, 132.9, 132.0, 130.1, 129.2, 121.1, 128.7,128.6, 127.0, 126.5, 84.3, 81.0, 79.0, 76.3, 75.4, 75.2, 75.0, 72.2,72.0, 58.4, 52.7, 45.5, 43.1, 40.1, 35.5, 26.7, 22.6, 22.0, 20.7, 14.7,9.5 ppm.

Linkage of Taxol to Delivery-Enhancing Transporter

The peptide (47.6 mg, 22.4 μmol) was dissolved in DMF (1.0 mL) under anN₂-atmosphere. DIEA (2.8 mg, 22.4 μmol) was added. A solution oftaxol-2′-chloroacetate (20.8 mg, 22.4 μmol) in DMF (1.0 mL) was added.Stirring at room temperature was continued for 6 hours. Water containing0.1% TFA (1.0 mL) was added, the sample was frozen and the solvents werelyophilized. The crude material was purified by RP-HPLC (eluent:water/MeCN*0.1% TFA: 85%-15%). A schematic of this reaction is shown inFIG. 18.

Synthesis of Related Conjugates

Using the conjugation conditions outlined above, the three additionalconjugates shown in were synthesized.

Cytotoxicity Assay

The taxol conjugates were tested for cytotoxicity in a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide (MTT) dyereduction. Results, which are shown in FIG. 20, demonstrate that thetaxol conjugated to r7 with a readily pH-releasable linker (CG 1062;R═Ac in the structure shown in FIG. 19) is significantly more cytotoxicthan either taxol alone or taxol conjugated to r7 with a less-readilypH-releasable linker (CG 1040; R═H in the structure shown in FIG. 19).

Example 5 Structure-Function Relationships of Fluorescently-LabeledPeptides Derived from Tat₄₉₋₅₇ Methods

General.

Rink amide resin and Boc₂O were purchased from Novabiochem.Diisopropylcarbodiimide, bromoacetic acid, fluorescein isothiocyanate(FITC-NCS), ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane,1,6-diaminohexane, trans-1,6-diaminocyclohexane, andpyrazole-1-carboxamidine were all purchased from Aldrich®. All solventsand other reagents were purchased from commercial sources and usedwithout further purification. The mono-Boc amines were synthesized fromthe commercially available diamines using a literature procedure (10equiv. of diamine and 1 equiv. of Boc₂O in chloroform followed by anaqueous work up to remove unreacted diamine) (34).N-tert-butoxycarbonyl-1,6-trans-diaminocyclohexane. Mp 159-161° C.; ¹HNMR (CDCl₃) δ 4.35 (br s, 1H), 3.37 (br s, 1H), 2.61 (br s, 1H),1.92-2.02 (m, 2H), 1.81-1.89 (m, 2H), 1.43 (s, 9H), 1.07-1.24 (m, 4H)ppm; ¹³C NMR (D₆-DMSO) δ 154.9, 77.3, 49.7, 48.9, 35.1, 31.4, 28.3 ppm;ES-MS (M+1) calcd 215.17. Found 215.22.

General Procedure for Peptide Synthesis.

Tat₄₉₋₅₇ (RKKRRQRRR), truncated and alanine-substituted peptides derivedfrom Tat₄₉₋₅₇ , Antennapedia ₄₃₋₅₈ (RQIKIWFQNRRMKWKK), and homopolymersof arginine (R5-R9) and d-arginine (r5-r9) were prepared with anautomated peptide synthesizer (ABI433) using standard solid-phase Fmocchemistry (35) with HATU as the peptide coupling reagent. Thefluorescein moiety was attached via a aminohexanoic acid spacer bytreating a resin-bound peptide (1.0 mmol) with fluoresceinisothiocyanate (1.0 mmol) and DIEA (5 mmol) in DMF (10 mL) for 12 h.Cleavage from the resin was achieved using 95:5 TFA/triisopropylsilane.Removal of the solvent in vacuo gave a crude oil which was trituratedwith cold ether. The crude mixture thus obtained was centrifuged, theether was removed by decantation, and the resulting orange solid waspurified by reverse-phase HPLC (H₂O/CH₃CN in 0.1% TFA). The productswere isolated by lyophilization and characterized by electrospray mass,spectrometry. Purity of the peptides was >95% as determined byanalytical reverse-phase HPLC (H₂O/CH₃CN in 0.1% TFA).

All peptides and peptoids synthesized contain an aminohexanoic (ahx)acid moiety attached to the N-terminal amino group with a fluoresceinmoiety (Fl) covalently linked to the amino group of the aminohexanoicacid spacer. The carboxyl terminus of every peptide and peptoid is acarboxamide.

Cellular Uptake Assay.

The arginine homopolymers and guanidine-substituted peptoids were eachdissolved in PBS buffer (pH 7.2) and their concentration was determinedby absorption of fluorescein at 490 nm (ε=67,000). The accuracy of thismethod for determining concentration was established by weighingselected samples and dissolving them in a known amount of PBS buffer.The concentrations determined by UV spectroscopy correlated with theamounts weighed out manually. Jurkat cells (human T cell line), murine Bcells (CH27), or human PBL cells were grown in 10% fetal calf serum andDMEM and each of these were used for cellular uptake experiments.Varying amounts of arginine and oligomers of guanidine-substitutedpeptoids were added to approximately 3×10⁶ cells in 2% FCS/PBS (combinedtotal of 200 μL) and placed into microtiter plates (96 well) andincubated for varying amounts of time at 23° C. or 4° C. The microtiterplates were centrifuged and the cells were isolated, washed with coldPBS (3×250 μL), incubated with 0.05% trypsin/0.53 mM EDTA at 37° C. for5 min, washed with cold PBS, and resuspended in PBS containing 0.1%propidium iodide. The cells were analyzed using fluorescent flowcytometry (FACScan, Becton Dickinson) and cells staining with propidiumiodide were excluded from the analysis. The data presented is the meanfluorescent signal for the 5000 cells collected.

Inhibition of Cellular Uptake with Sodium Azide.

The assays were performed as previously described with the exceptionthat the cells used were preincubated for 30 min with 0.5% sodium azidein 2% FCS/PBS buffer prior to the addition of fluorescent peptides andthe cells were washed with 0.5% sodium azide in PBS buffer. All of thecellular uptake assays were run in parallel in the presence and absenceof sodium azide.

Cellular Uptake Kinetics Assay.

The assays were performed as previously described except the cells wereincubated for 0.5, 1, 2, and 4 min at 4° C. in triplicate in 2% FCS/PBS(50 μl) in microtiter plates (96 well). The reactions were quenched bydiluting the samples into 2% FCS/PBS (5 mL). The assays were then workedup and analyzed by fluorescent flow cytometry as previously described.

Results

To determine the structural requirements for the cellular uptake ofshort arginine-rich peptides, a series of fluorescently-labeledtruncated analogues of Tat₄₉₋₅₇ were synthesized using standardsolid-phase chemistry. See, e.g., Atherton, E. et al. SOLID-PHASEPEPTIDE SYNTHESIS (IRL: Oxford, Engl. 1989). A fluorescein moiety wasattached via an aminohexanoic acid spacer on the amino termini. Theability of these fluorescently labeled peptides to enter Jurkat cellswas then analyzed using fluorescent activated cell sorting (FACS). Thepeptide constructs tested were Tat₄₉₋₅₇ (Fl-ahx-RKKRRQRRR): Tat₄₉₋₅₆(Fl-ahx-RKKRRQRR), Tat₄₉₋₅₅ (Fl-ahx-RKKRRQR), Tat₅₀₋₅₇(Fl-ahx-KKRRQRRR), and Tat₅₁₋₅₇ (Fl-ahx-KRRQRRR). Differentiationbetween cell surface binding and internalization was accomplishedthroughout by running a parallel set of assays in the presence andabsence of sodium azide. Because sodium azide inhibits energy-dependentcellular uptake but not cell surface binding, the difference influorescence between the two assays provided the amount of fluorescenceresulting from internalization.

Deletion of one arginine residue from either the amine terminus(Tat₅₀₋₅₇) or the carboxyl terminus (Tat₄₉₋₅₆) resulted in an 80% lossof intracellular fluorescence compared to the parent sequence(Tat₄₉₋₅₇). From the one amino acid truncated analogs, further deletionof R-56 from the carboxyl terminus (Tat₄₉₋₅₅) resulted in an additional60% loss of intracellular fluorescence, while deletion of K-50 from theamine terminus (Tat₅₁₋₅₇) did not further diminish the amount ofinternalization. These results indicate that truncated analogs ofTat₄₉₋₅₇ are significantly less effective at the transcellular deliveryof fluorescein into Jurkat cells, and that the arginine residues appearto contribute more to cellular uptake than the lysine residues.

To determine the contribution of individual amino acid residues tocellular uptake, analogs containing alanine substitutions at each siteof Tat₄₉₋₅₇ were synthesized and assayed by FACS analysis (FIG. 22). Thefollowing constructs were tested: A-49 (Fl-ahx-AKKRRQRRR), A-50(Fl-ahx-RAKRRQRRR), A-51 (Fl-ahx-RKARRQRRR), A-52 (Fl-ahx-RKKARQRRR),A-53 (Fl-ahx-RKKRAQRRR), A-54 (Fl-ahx-RKKRRARRR), A-55(Fl-ahx-RKKRRQARR), A-56 (Fl-ahx-RKKRRQRAR), and A-57(Fl-ahx-RKKRRQRRA). Substitution of the non-charged glutamine residue ofTat₄₉₋₅₇ with alanine (A-54) resulted in a modest decrease in cellularinternalization. On the other hand, alanine substitution of each of thecationic residues individually produced a 70-90% loss of cellularuptake. In these cases, the replacement of lysine (A-50, A-51) orarginine (A-49, A-52, A-55, A-56, A-57) with alanine had similar effectsin reducing uptake.

To determine whether the chirality of the transporter peptide wasimportant, the corresponding d-(d-Tat₄₉₋₅₇), retro-l-(Tat₅₇₋₄₉), andretro-inverso isomers (d-Tat₅₇₋₄₉) were synthesized and assayed by FACSanalysis (FIG. 23). Importantly, all three analogs were more effectiveat entering Jurkat cells then Tat₄₉₋₅₇. These results indicated that thechirality of the peptide backbone is not crucial for cellular uptake.Interestingly, the retro-1 isomer (Tat₅₇₋₄₉) which has three arginineresidues located at the amine terminus instead of one arginine and twolysines found in Tat₄₉₋₅₇ demonstrated enhanced cellular uptake. Thus,residues at the amine terminus appear to be important and that argininesare more effective than lysines for internalization. The improvedcellular uptake of the unnatural d-peptides is most likely due to theirincreased stability to proteolysis in 2% FCS (fetal calf serum) used inthe assays. When serum was excluded, the d- and l-peptides wereequivalent as expected.

These initial results indicated that arginine content is primarilyresponsible for the cellular uptake of Tat₄₉₋₅₇. Furthermore, theseresults were consistent with our previous results where we demonstratedthat short oligomers of arginine were more effective at entering cellsthen the corresponding short oligomers of lysine, ornithine, andhistidine. What had not been established was whether argininehomo-oligomers are more effective than Tat₄₉₋₅₇. To address this point,Tat₄₉₋₅₇ was compared to the l-arginine (R5-R9) and d-arginine (r5-r9)oligomers. Although Tat₄₉₋₅₇ contains eight cationic residues, itscellular internalization was between that of R6 and R7 (FIG. 24)demonstrating that the presence of six arginine residues is the mostimportant factor for cellular uptake. Significantly, conjugatescontaining 7-9 arginine residues exhibited better uptake than Tat₄₉₋₅₇.

To quantitatively compare the ability of these arginine oligomers andTat₄₉₋₅₇ to enter cells, Michaelis-Menton kinetic analyses wereperformed. The rates of cellular uptake were determined after incubation(3° C.) of the peptides in Jurkat cells for 30, 60, 120, and 240 seconds(Table 1). The resultant K_(m) values revealed that r9 and R9 enteredcells at rates approximately 100-fold and 20-fold faster than Tat₄₇₋₅₉respectively. For comparison, Antennapedia ₄₃₋₅₈ was also analyzed andwas shown to enter cells approximately 2-fold faster than Tat₄₇₋₅₉, butsignificantly slower than r9 or R9.

TABLE 1 Michaelis-Menton kinetics: Antennapedia₄₃₋₅₈(Fl-ahx-RQIKIWFQNRRMKWKK). peptide K_(m)(μM) V_(max) Tat₄₉₋₅₇ 770 0.38Antennapedia₄₃₋₅₈ 427 0.41 R9 44 0.37 r9 7.6 0.38

Example 6

Design and Synthesis of Peptidomimetic Analogs of Tat₄₉₋₅₇

Methods

General Procedure for Peptoid Polyamine Synthesis.

Peptoids were synthesized manually using a flitted glass apparatus andpositive nitrogen pressure for mixing the resin following the literatureprocedure developed by Zuckermann. See, e.g., Murphy, J. E. et al.,Proc. Natl. Acad. Sci. USA 95, 1517-1522 (1998); Simon, R. J. et al.,Proc. Natl. Acad. Sci. USA 89, 9367-9371 (1992); Zuckermann, R. N. etal., J. Am. Chem. Soc. 114, 10646-10647 (1992). Treatment ofFmoc-substituted Rink amide resin (0.2 mmol) with 20% piperidine/DMF (5mL) for 30 min (2×) gave the free resin-bound amine which was washedwith DMF (3×5 mL). The resin was treated with a solution of bromoaceticacid (2.0 mmol) in DMF (5 mL) for 30 min. This procedure was repeated.The resin was then washed (3×5 mL DMF) and treated with a solution ofmono-Boc diamine (8.0 mmol) in DMF (5 mL) for 12 hrs. These two stepswere repeated until an oligomer of the required length was obtained(Note: the solution of mono-Boc diamine in DMF could be recycled withoutappreciable loss of yield). The resin was then treated withN-Fmoc-aminohexanoic acid (2.0 mmol) and DIC (2.0 mmol) in DMF for 1 hand this was repeated. The Fmoc was then removed by treatment with 20%piperidine/DMF (5 mL) for 30 min. This step was repeated and the resinwas washed with DMF (3×5 mL). The free amine resin was then treated withfluorescein isothiocyanate (0.2 mmol) and DIEA (2.0 mmol) in DMF (5 mL)for 12 hrs. The resin was then washed with DMF (3×5 mL) anddichloromethane (5×5 mL). Cleavage from the resin was achieved using95:5 TFA/triisopropylsilane (8 mL). Removal of the solvent in vacuo gavea crude oil which was triturated with cold ether (20 mL). The crudemixture thus obtained was centrifuged, the ether was removed bydecantation, and the resulting orange solid was purified byreverse-phase HPLC (H₂O/CH₃CN in 0.1% TFA). The products were isolatedby lyophilization and characterized by electrospray mass spectrometryand in selected cases by ¹H NMR spectroscopy.

General Procedure for Perguanidinylation of Peptoid Polyamines.

A solution of peptoid amine (0.1 mmol) dissolved in deionized water (5mL) was treated with sodium carbonate (5 equivalents per amine residue)and pyrazole-1-carboxamidine (5 equivalents per amine residue) andheated to 50° C. for 24-48 hr. The crude mixture was then acidified withTFA (0.5 mL) and directly purified by reverse-phase HPLC (H₂O/CH₃CN in0.1% TFA). The products were characterized by electrospray massspectrometry and isolated by lyophilization and further purified byreverse-phase HPLC. The purity of the guanidine-substituted peptoidswas >95% as determined by analytical reverse-phase HPLC(H₂O/CH₃CN in0.1% TFA).

Results

Utilizing the structure-function relationships that had been determinedfor the cellular uptake of Tat₄₇₋₅₉, we designed a set of polyguanidinepeptoid derivatives that preserve the 1,4 backbone spacing of sidechains of arginine oligomers, but have an oligo-glycine backbone devoidof stereogenic centers. These peptoids incorporating arginine-like sidechains on the amide nitrogen were selected because of their expectedresistance to proteolysis, and potential ease and significantly lowercost of synthesis (Simon et al., Proc. Natl. Acad. Sci. USA 89:9367-9371(1992); Zuckermann, et al., J. Am. Chem. Soc. 114:10646-10647 (1992).Furthermore, racemization, frequently encountered in peptide synthesis,is not a problem in peptoid synthesis; and the “sub-monomer” peptoidapproach allows for facile modification of side-chain spacers. Althoughthe preparation of an oligurea and peptoid-peptide hybrid (Hamy, et al,Proc. Natl. Acad. Sci. USA 94:3548-3553 (1997)) derivatives of Tat₄₉₋₅₇have been previously reported, their cellular uptake was not explicitlystudied.

The desired peptoids were prepared using the “sub-monomer” approach(Simon et al.; Zuckermann et al.) to peptoids followed by attachment ofa fluorescein moiety via an aminohexanoic acid spacer onto the aminetermini. After cleavage from the solid-phase resin, the fluorescentlylabeled polyamine peptoids thus obtained were converted in good yields(60-70%) into polyguanidine peptoids by treatment with excesspyrazole-1-carboxamidine (Bernatowicz, et al., J. Org. Chem.57:2497-2502 (1992) and sodium carbonate (as shown in FIG. 25).Previously reported syntheses of peptoids containing isolated N-Argunits have relied on the synthesis of N-Arg monomers (5-7 steps) priorto peptoid synthesis and the use of specialized and expensive guanidineprotecting groups (Pmc, Pbf) (Kruijtzer, et al., Chem. Eur. J.4:1570-1580 (1998); Heizmann, et al. Peptide Res. 7:328-332 (1994). Thecompounds reported here represent the first examples ofpolyguanidinylated peptoids prepared using a perguanidinylation step.This method provides easy access to polyguanidinylated compounds fromthe corresponding polyamines and is especially useful for the synthesisof perguanidinylated homooligomers. Furthermore, it eliminates the useof expensive protecting groups (Pbf, Pmc). An additional example of aperguanidinylation of a peptide substrate using a noveltriflyl-substituted guanylating agent has recently been reported(Feichtinger, et al., J. Org. Chem. 63:8432-8439 (1998)).

The cellular uptake of fluorescently labeled polyguanidine N-arg-5,7,9peptoids was compared to the corresponding d-arginine peptides r-5,7,9(similar proteolytic properties) using Jurkat cells and FACS analysis.The amount of fluorescence measured inside the cells with N-arg-5,7,9was proportional to the number of guanidine residues:N-arg9>N-arg7>N-arg5 (FIG. 26), analogous to that found for r-5,7,9.Furthermore, the N-arg-5,7,9 peptoids showed only a slightly loweramount of cellular entry compared to the corresponding peptides,r-5,7,9. The results demonstrate that the hydrogen bonding along thepeptide backbone of Tat₄₉₋₅₇ or arginine oligomers is not a requiredstructural element for cellular uptake and oligomericguanidine-substituted peptoids can be utilized in place of arginine-richpeptides as molecular transporters. The addition of sodium azideinhibited internalization demonstrating that the cellular uptake ofpeptoids was also energy dependent.

Example 7 The Effect of Side Chain Length on Cellular Uptake

After establishing that the N-arg peptoids efficiently crossed cellularmembranes, the effect of side chain length (number of methylenes) oncellular uptake was investigated. For a given number of guanidineresidues (5,7,9), cellular uptake was proportional to side chain length.Peptoids with longer side chains exhibited more efficient cellularuptake. A nine-mer peptoid analog with a six-methylene spacer betweenthe guanidine head groups and the backbone (N-hxg9) exhibited remarkablyhigher cellular uptake than the corresponding d-arginine oligomer (r9).The relative order of uptake was N-hxg9 (6 methylene)>N-btg9 (4methylene)>r9 (3 methylene)>N-arg9 (3 methylene)>N-etg9 (2 methylene)(FIG. 27). Of note, the N-hxg peptoids showed remarkably high cellularuptake, even greater than the corresponding d-arginine oligomers. Thecellular uptake of the corresponding heptamers and pentamers also showedthe same relative trend. The longer side chains embodied in the N-hxgpeptoids improved the cellular uptake to such an extent that the amountof internalization was comparable to the corresponding d-arginineoligomer containing one more guanidine residue (FIG. 28). For example,the N-hxg7 peptoid showed comparable cellular uptake to r8.

To address whether the increase in cellular uptake was due to theincreased length of the side chains or due to their hydrophobic nature,a set of peptoids was synthesized containing cyclohexyl side chains.These are referred to as the N-chg-5,7,9 peptoids. These contain thesame number of side chain carbons as the N-hxg peptoids but possessdifferent degrees of freedom. Interestingly, the N-chg peptoid showedmuch lower cellular uptake activity than all of the previously assayedpeptoids, including the N-etg peptoids (FIG. 29). Therefore, theconformational flexibility and sterically unencumbered nature of thestraight chain alkyl spacing groups is important for efficient cellularuptake.

Discussion

The nona-peptide, Tat₄₉₋₅₇, has been previously shown to efficientlytranslocate through plasma membranes. The goal of this research was todetermine the structural basis for this effect and use this informationto develop simpler and more effective molecular transporters. Towardthis end, truncated and alanine substituted derivatives of Tat₄₉₋₅₇conjugated to a fluoroscein label was prepared. These derivativesexhibited greatly diminished cellular uptake compared to Tat₄₉₋₅₇,indicating that all of the cationic residues of Tat₄₉₋₅₇ are requiredfor efficient cellular uptake. When compared with our previous studieson short oligomers of cationic oligomers, these findings suggested thatan oligomer of arginine might be superior to Tat₄₉₋₅₇ and certainly moreeasily and cost effectively prepared. Comparison of short arginineoligomers with Tat₄₉₋₅₇ showed that members of the former were indeedmore efficiently taken into cells. This was further quantified for thefirst time bt Michaelis-Menton kinetics analysis which showed that theR9 and r9 oligomers had Km values 30-fold and 100-fold greater than thatfound for Tat₄₉₋₅₇.

Given the importance of the guanidino head group and the apparentinsensitivity of the oligomer chirality revealed in our peptide studies,we designed and synthesized a novel series of polyguanidine peptoids.The peptoids N-arg-5,7,9, incorporating the arginine side chain,exhibited comparable cellular uptake to the corresponding d-argininepeptides r-5,7,9, indicating that the hydrogen bonding along the peptidebackbone and backbone chirality are not essential for cellular uptake.This observation is consistent with molecular models of these peptoids,arginine oligomers, and Tat₄₉₋₅₇ all of which have a deeply embeddedbackbone and a guanidinium dominated surface. Molecular models furtherreveal that these structural characteristics are retained in varyingdegree in oligomers with different alkyl spacers between the peptoidbackbone and guanidino head groups. Accordingly, a series of peptoidsincorporating 2-(N-etg), 4-(N-btg), and 6-atom (N-hxg) spacers betweenthe backbone and side chain were prepared and compared for cellularuptake with the N-arg peptoids (3-atom spacers) and d-arginineoligomers. The length of the side chains had a dramatic affect oncellular entry. The amount of cellular uptake was proportional to thelength of the side chain with N-hxg>N-btg>N-arg>N-etg. Cellular uptakewas improved when the number of alkyl spacer units between the guanidinehead group and the backbone was increased. Significantly, N-hxg9 wassuperior to r9, the latter being 100-fold better than Tat₄₉₋₅₇. Thisresult led us to prepare peptoid derivatives containing longer octylspacers (N-ocg) between the guanidino groups and the backbone. Issuesrelated to solubility prevented us from testing these compounds.

Because both perguanidinylated peptides and perguanidinylated peptoidsefficiently enter cells, the guanidine head group (independent ofbackbone) is apparently the critical structural determinant of cellularuptake. However, the presence of several (over six) guanidine moietieson a molecular scaffold is not sufficient for active transport intocells as the N-chg peptoids did not efficiently translocate into cells.Thus, in addition to the importance of the guanidine head group, thereare structure/conformational requirements that are significant forcellular uptake.

In summary, this investigation identified a series of structuralcharacteristics including sequence length, amino acid composition, andchirality that influence the ability of Tat₄₉₋₅₇ to enter cells. Thesecharacteristics provided the blueprint for the design of a series ofnovel peptoids, of which 17 members were synthesized and assayed forcellular uptake. Significantly, the N-hxg9 transporter was found to besuperior in cell uptake to r9 which was comparable to N-btg9. Hence,these peptoid transporters proved to be substantially better thanTat₄₉₋₅₇. This research established that the peptide backbone andhydrogen bonding along that backbone are not required for cellularuptake, that the guanidino head group is superior to other cationicsubunits, and most significantly, that an extension of the alkyl chainbetween the backbone and the head group provides superior transporters.In addition to better uptake performance, these novel peptoids offerseveral advantages over Tat₄₉₋₅₇ including cost-effectiveness, ease ofsynthesis of analogs, and protease stability. These features along withtheir significant water solubility (>100 mg/mL) indicate that thesenovel peptoids could serve as effective transporters for the moleculardelivery of drugs, drug candidates, and other agents into cells.

Example 8 Synthesis of Itraconazole-Transporter Conjugate

This Example provides one application of a general strategy forattaching a delivery-enhancing transporter to a compound that includes atriazole structure. The scheme, using attachment of itraconazole to anarginine (r7) delivery-enhancing transporter as an example, is shown inFIG. 30. In the scheme, R is H or alkyl, n is 1 or 2, and X is ahalogen.

The reaction involves making use of quaternization of a nitrogen in thetriazole ring to attach an acyl group that has a halogen (e.g., Br, Fl,I) or a methyl ester. Compound 3 was isolated by HPLC. Proton NMR in D₂Orevealed itraconazole and transporter peaks.

The methyl ester provided yields of 70% and greater, while yieldsobtained using the Br-propionic acid/ester pair were 40-50%. The acylderivative is then reacted with the amine of the delivery-enhancingtransporter to form the conjugate. Alternatively, the halogenated acylgroup can first be attached to the transporter molecule through an amidelinkage, after which the reaction with the drug compound is conducted.

Example 9 Preparation of FK506 Conjugates

This Example describes the preparation of conjugates in which FK506 isattached to a delivery-enhancing transporter. Two different linkers wereused, each of which released FK506 at physiological pH (pH 5.5 to 7.5),but had longer half-lives at more acidic pH. These schemes arediagrammed in FIGS. 31A and B.

Linker 1: 6-Maleimidocaproic Hydrazide Trifluoroacetate (Scheme I andII)

A solution of FK506 (1) (0.1 g, 124.4 μmol), 6-maleiimidocaproichydrazide trifluoroacetate (2) (0.126 g, 373.2 μmol) and trifluoroaceticacid (catalytic, 1 μL) in anhydrous methanol (5 mL) was stirred at roomtemperature for 36 h. The reaction was monitored by thin layerchromatography that showed almost complete disappearance of the startingmaterial. [TLC solvent system—dichloromethane (95): methanol (5),R_(f)=0.3]. The reaction mixture was concentrated to dryness anddissolved in ethyl acetate (20 mL). The organic layer was washed withwater and 10% sodium bicarbonate solution and then dried over sodiumsulfate, filtered and concentrated. The residue was purified by columnchromatography using dichloromethane (96): methanol (4) as eluent togive the hydrazone 3 (0.116 g, 92%).

A solution of the above hydrazone (3) (0.025 g, 24.7 μmol), transporter(1×, Bacar₉CCONH₂.9TFA, Bacar₇CCONH₂.7TFA, BacaCCONH₂, NH₂r₇CCONH₂.8TFA,NH₂R₇CCONH₂.8TFA) and diisopropylethylamine (1×) in anhydrousdimethylformamide (1 mL) were stirred under nitrogen at room temperaturefor 36 h when TLC indicated the complete disappearance of the startinghydrazone. Solvent was evaporated from the reaction mixture and theresidue purified by reverse phase HPLC using trifluoroacetic acidbuffered water and acetonitrile.

Yields of conjugates with various transporters:

-   -   Conjugate with Bacar₉CCONH₂.9TFA (4)-73%    -   Bacar₇CCONH₂.7TFA (5)-50%    -   BacaCCONH₂ (6)-52.9%    -   NH₂r₇CCONH₂.8TFA (7)-43.8%    -   NH₂R₇CCONH₂.8TFA (8)-62.8%

Structures of all the products were confirmed by ¹H-NMR spectra and TOFMS analysis.

Linker 2: 2-(2-pyridinyldithio) ethyl hydrazine carboxylate (Scheme IIIand IV)

A solution of FK506 (1) (0.1 g, 124.4 μl), 2-(2-pyridinyldithio) ethylhydrazine carboxylate (9) (0.091 g, 373.2 μmol) and trifluoroacetic acid(catalytic, 1 μL) in anhydrous methanol (5 mL) was stirred at roomtemperature for 16 h. The reaction was monitored by thin layerchromatography that showed almost complete disappearance of the startingmaterial. [TLC solvent system−ethyl acetate R_(f)=0.5]. The reactionmixture was concentrated to dryness and dissolved in ethyl acetate (20mL). The organic layer was washed with water and 10% sodium bicarbonatesolution and then dried over sodium sulfate, filtered and concentrated.The residue was purified by column chromatography using dichloromethane(97): methanol (3) as eluent to give the hydrazone 10 (0.091 g, 71%)

Example 10

This example illustrates the conjugation of cyclosporin to a transportmoiety using a pH sensitive linking group (see FIGS. 6A and 9B).

In this example, cyclosporin is converted to its α-chloroacetate esterusing chloroacetic anhydride to provide 61 (see FIG. 6). The ester 61 isthen treated with benzylamine to provide 6ii. Reaction of the amine withBoc-protected iminodiacetic acid anhydride provides the acid 6iii whichis then converted to an activated ester (6iv) with N-hydroxysuccinimide. Coupling of 61v with L-Arginine heptamer provides theBOC-protected conjugate 6v, which can be converted to conjugate 6vi byremoval of the BOC protecting group according to established methods.

Transport moieties having arginine groups separated by, for example,glycine, ε-aminocaproic acid, or γ-aminobutyric acid can be used inplace of the arginine heptamer in this and in the following examplesthat show oligoarginine transport groups.

Example 11

This example illustrates the conjugation of acyclovir to a transportmoiety.

a. Conjugation of acyclovir to r₇CONH₂

This example illustrates the conjugation of acyclovir to r₇CONH₂ via thelinking group:

i) Preparation of acyclovir α-chloroester:

A solution of acyclovir (100 mg, 0.44 mmol), dimethylaminopyridine (5.4mg, 0.044 mmol) and chloroacetic anhydride (226 mg, 1.32 mmol) indimethylformamide (9 mL) was stirred at room temperature for 18 h. Thedimethylformamide was removed by evaporation. The crude product waspurified by reverse-phase HPLC (22 mm×250 mm C-18 column, a 5-25%CH₃CN/H₂O gradient with 0.1% trifluoroacetic acid, 214 and 254 nm UVdetection) and lyophilized. The product was obtained as a white powder(62 mg, 47%). ¹H NMR (300 MHz, DMSO-d₆) δ 10.67 (s, 1H), 7.88 (s, 1H),6.53 (s, 1H), 5.27 (s, 2H), 4.35 (s, 2H), 4.21 (t, J=3 Hz, 2H), 3.70 (t,J=3 Hz, 2H); ¹³C NMR (75 MHz, DMSO-d₆) δ 168.1, 157.6, 154.8, 152.3,138.6, 117.1, 72.7, 67.1, 65.2, 41.8; TOF-MS (m/z): 302.0 [M+H].

ii) Conjugation of Acyclovir a-chloro ester to H₂N—C-r7-CONH₂

A solution of acyclovir α-chloroester (7 mg, 0.024 mmol), H₂N—C-r7-CONH₂(50 mg, 0.024 mmol) and diisopropylethylamine (6.4 μL, 0.036 mmol) indimethylformamide (1 mL) was stirred for 18 h. The dimethylformamide wasremoved by evaporation. The crude product was purified by reverse-phaseHPLC (22 mm×250 mm C-18 column, a 5-25% CH₃CN/H₂O gradient with 0.1%trifluoroacetic acid, 214 and 254 nm UV detection) and lyophilized. Thedesired product was obtained as a white powder (24 mg, 69%). TOF-MS(m/z): 494.6 [(M+H)/3], 371.0 [(M+H)/4].

The yield could be increased by using 10 molar equivalents ofdiisopropylethylamine rather than 1.5 molar equivalents. Product wasagain obtained as a white powder (79%). TOF-MS (m/z): 508.7 [(M+H)/3],381.5 [(M+H)/4], 305.5 [(M+H)/5].

b. Conjugation of Acyclovir to a Biotin-Containing Derivative ofr₅-Cys-CONH₂

Reactions were carried out as illustrated above, using the synthetictechniques provided in the examples above.

i) Biotin-aminocaproic acid-r5-Cys(acyclovir)-CONH₂ was obtained as awhite powder (36%). TOF-MS (m/z): 868.2 {(M+2 TFA)/2], 811.2 [(M+1TFA)/2], 754.1 [(M+1 TFA)/3], 503.0 [(M+H)/3], 377.4 [(M+H)/4].

Similarly,

ii) Biotin-aminocaproic acid-r7-C(acyclovir)-CONH₂— was obtained as awhite powder (33%). TOF-MS (m/z):722.1 [(M+3 TFA)/3], 684.6 [(M+2TFA)/3], 607.1 [(M+H)/3], 455.5 [(M+H)/4], 364.8 [(M+H)/5], 304.3[(M+H)/6].

Example 12

This example illustrates the conjugation of hydrocortisone to atransport moiety.

a. Conjugation of hydrocortisone to r₇CONH₂

i) Preparation of hydrocortisone a-chloroester:

To a solution of hydrocortisone (500 mg, 1.38 mmol), scandium triflate(408 mg, 0.83 mmol) and chloroacetic anhydride (708 mg, 4.14 mmol) indry THF was added dimethylaminopyridine (506 mg, 4.14 mmol). Thesolution turned bright yellow upon addition of dimethylaminopyridine.After 30 min the solvent was evaporated off and the crude material takenup into ethyl acetate (100 mL). The ethyl acetate layer was washed with1.0 N HCl and brine. The organic phase was collected, dried (Na₂SO₄) andevaporated to provide the product as a white solid (533 mg, 88%). ¹H NMR(300 MHz, DMSO-d₆) δ 5.56 (s, 1H), 5.46 (s, 1H), 5.20 (d, J=18 Hz, 1H),4.85 (d, J=18 Hz, 1H), 4.51 (s, 2H), 4.37 (br s, 1H), 4.27 (br s, 1H),2.54-2.33 (m, 2H), 2.22-2.03 (m, 3H), 1.99-1.61 (m, 8H), 1.52-1.24 (m,5H), 1.02-0.98 (d, J=12 Hz, 1H), 0.88-0.85 (d, J=9 Hz, 1H), 0.77 (s,3H); ¹³C NMR (75 MHz, DMSO-d₆) δ 205.4, 198.8, 173.0, 167.6, 122.3,89.5, 69.7, 67.3, 56.4, 52.4, 47.8, 41.6, 39.7, 35.0, 34.3, 34.0, 33.6,32.3, 32.0, 24.2, 21.3, 17.4; TOF-MS (m/z): 439.1 (M+H).

(Reference for acetylation—Zhao, H.; Pendri, A.; Greenwald, R. B. J.Org. Chem. 1998, 63, 7559-7562.)

ii) Coupling to R′NH-Cys-r₇-CONH₂

A solution of hydrocortisone α-chloroester (31 mg, 0.071 mmol),H₂N—C-r7-CONH₂ (150 mg, 0.071 mmol) and diisopropylethylamine (15 μL,0.085 mmol) in dimethylformamide (1 mL) was stirred for 18 h. Thedimethylformamide was evaporated off. The crude product purified byreverse-phase HPLC (22 mm×250 mm C-18 column, a 5-30% CH₃CN/H₂O gradientwith 0.1% trifluoroacetic acid, 214 and 254 nm UV detection) andlyophilized. The desired product was obtained as a white powder (25 mg,14%). TOF-MS (m/z): 1037.4 [(M+4 TFA)/2], 616.1 [(M+2 TFA)/3], 578.3[(M+1 TFA)/3], 540.5 [(M+H)/3], 405.7 [(M+H)/4], 324.5 [(M+H)/5].

The use of 10 molar equivalents of diisopropylethylamine rather than 1.2molar equivalents provided the desired product as a yellow powder (52%yield). TOF-MS (m/z): 887.0 [(M+1TFA)/2], 830.6 [(M+H)/2], 553.7[(M+H)/3], 415.5 [(M+H)/4].

b. Conjugation of Hydrocortisone to a Biotin-Containing Derivative ofr₅-Cys-CONH₂

Reactions were carried out as illustrated above, using the synthetictechniques provided in the examples above.

i) Biotin-Aminocaproic Acid-r5-C(hydrocortisone)-CONH₂—

Used 10 molar equivalents of diisopropylethylamine rather than 1.2 molarequivalents. Product a white powder (65%). TOF-MS (m/z): 880.7 [(M+1TFA)/2], 548.7 [(M+H)/3].

ii) Biotin-Aminocaproic Acid-r7-C(hydrocortisone)-CONH₂—

Used 10 molar equivalents of diisopropylethylamine rather than 1.2 molarequivalents. Product a white powder (36%). TOF-MS (m/z): 692.3 [(M+1TFA)/3], 652.8 [(M+H)/3], 520.0 [(M+1 TFA)/4], 490.0 [(M+H)/4], 392.5[(M+H)/5].

Example 13

This example illustrates the conjugation of taxol to a transport moiety.

a. Conjugation of Taxol to rγ-CONH₂

This example illustrates the application of methodology outlined aboveto the preparation of a taxol conjugate (see FIG. 12).

i) Preparation of a taxol a-chloroacetate ester

Taxol was treated with α-chloro acetic anhydride providing the C-2′chloro acetyl derivative 12i in essentially quantitative yield.

ii) Formation of Taxol Conjugate

The halogen atom of the chloroacetate ester was displaced by the thiolof an N-terminal (L) cysteine containing heptamer of arginine. To avoiddegradation of the transporter entity by proteases in-vivo, D-argininewas used as the building unit. Conjugation reactions were performed atroom temperature in DMF in the presence of diisopropylethylamine. Thefinal products were isolated by RP-HPLC and lyophilized to whitepowders. It is important to note that the native conjugate (R═H) isisolated as its TFA salt at the cysteine primary amine. The conjugatesare generally quite hygroscopic and readily dissolve in water.

The conjugate wherein R═H was designed to release the parent drug via anucleophilic attack of the N-terminal nitrogen onto the C2′ estercarbonyl. The protonation state of this nitrogen is crucial for thismechanism, since only the free amine will be capable of this release.Additionally, both conjugates share a common α-hetero atom substitutedacetate moiety making them susceptible to simple ester hydrolysis. Thisoffers an additional release pathway.

Example 14

This example illustrates two methods of linking active agents totransport moieties. Illustration is provided for retinoic acidderivatives linked to poly-D-Arg derivatives but can be applied tolinkages between other biological agents and the transport moieties ofthe present invention.

A. Linkage Between a Biological Agent Having an Aldehyde FunctionalGroup

This example illustrates the preparation of a conjugate between anonamer of D-arginine (H₂N-r₉-CO₂H.10TFA) and either all trans-retinalor β-cis-retinal. FIG. 33 provides a schematic presentation of thereactions. As seen in FIG. 33, condensation of either retinal withH₂N-r₉-CO₂H.10TFA in MeOH in the presence of 4 Å molecular seives atroom temperature for four hours results in the formation of a Schiffbase-type linkage between the retinal aldehyde and the amino terminalgroup. Purification of the conjugate can be accomplished by filteringthe molecular sieves and removing methanol under reduced pressure.

b. Conjugation of Retinoic Acid to rγ-CONH₂

This example illustrates the preparation of a conjugate between retinoicacid and rγ-CONH₂ using the linking group

Here, preparation of the conjugate follows the scheme outlined in FIG.34. In this scheme, retinoic acid (34ii) is first combined with thechloroacetate ester of 4-hydroxymethyl-2,6-dimethylphenol (34i) toprovide the conjugate shown as 34iii. Combination of 34i with retinoicacid in methylene chloride in the presence of dicyclohexylcarbodiimideand a catalytic amount of 4-dimethylaminopyridine provided the retinoidderivative 34iii in 52-57% yield. Condensation of 34iii withH₂NCys-r₇CONH₂.8TFA in the presence of diisopropylethylamine (DMF, roomtemperature, 2 h) provides the desired conjugated product 34iv.

Example 15 Synthesis of Cyclosporin Conjugated to a BiotinylatedPentamer, Heptamer, and Nonamer of D-arginine Methods

A. Linking Cyclosporin to Delivery-Enhancing Transporters

1. Preparation of the α-Chloroacetyl Cyclosporin A Derivative.

The α-chloroacetyl cyclosporin A derivative was prepared as shown inFIG. 1. Cyclosporin A (152.7 mg, 127 μmol) and chloroacetic acidanhydride (221.7 mg; 1300 μmol) were placed into a dry flask underN₂-atmosphere. Pyridine (1.0 mL) was added and the solution was heatedto 50° C. (oil bath). After 16 hours the reaction was cooled to roomtemperature and quenched with water (4.0 mL). The resulting suspensionwas extracted with diethylether (Σ15 mL). The combined organic layerswere dried over MgSO₄. Filtration and evaporation of solvents in vacuodelivered a yellow oil, which was purified by flash chromatography onsilica gel (eluent: EtOAc/hexanes: 40%-80%) yielding 136 mg (106.4 mmol,83%) of the desired product.

2. Coupling to Transporter Molecules

A general procedure for the coupling of cysteine containing peptides tothe α-chloro acetyl Cyclosporin A derivative is shown in FIG. 2.

a. Labeled Peptides

The cyclosporin A derivative and the labeled peptide (1 equivalent) weredissolved in DMF (˜10 mmol of Cyclosporin A derivative/mL DMF) under anN₂-atmosphere. Diisopropylethylamine (10 equivalents) was added andstirring at room temperature was continued until all starting materialwas consumed (usually after 16 hours) (FIG. 3). The solvents wereremoved in vacuo and the crude reaction product was dissolved in waterand purified by reversed phase high pressure liquid chromatography(RP-HPLC) (eluent: water/MeCN*TFA). The products were obtained in thefollowing yields:

B-aca-r5-Ala-Ala-Cys-O-acyl-Cyclosporin A: 47%

B-aca-r7-Cys-O-acyl-Cyclosporin A: 43%

B-aca-r9-Cys-O-acyl-Cyclosporin A: 34%

B-aca-Cys-O-acyl-Cyclosporin A: 55%

b. Unlabeled Peptides

The peptide (34.7 mg, 15.3 μl) and the Cyclosporin A derivative (19.6mg, 15.3 μmol) were dissolved in DMF (1.0 mL) under an N₂-atmosphere(FIG. 4). Diisopropylethylamine (19.7 mg, 153 μmol) was added andstirring at room temperature was continued. After 12 hours the solventwas removed in vacuo. The crude material was dissolved in water andpurified by RP-HPLC (eluent: water/MeCN*TFA) yielding the pure product(24.1 mg, 6.8 mmol, 44%).

Example 16 Preparation of Hydrocortisone Conjugated to a BiotinylatedPentamer, Heptamer, and Nonamer of D-Arginine Methods

A. Linking of Hydrocortisone to Delivery-Enhancing Transporters

Step 1—Acylation of Hydrocortisone with Chloroacetic Anhydride.

A solution of hydrocortisone (200 mg, 0.55 mmol) and chloroaceticanhydride (113 mg, 0.66 mmol) in pyridine (5 mL) was stirred at roomtemperature for 2 h (FIG. 10). The solvent was evaporated off and thecrude product was chromatographed on silica using 50% hexanes/ethylacetate as the eluent. Product isolated a whites solid (139 mg, 58%).

Step 2—Linking to Transporter.

A solution of the chloroacetic ester of hydrocortisone (0.0137 mmol), atransporter containing a cysteine residue (0.0137) anddiisopropylethylamine (DIEA) (0.0274 mmol) in dimethylformamide (DMF) (1mL) was stirred at room temperature for 18 h (FIG. 11). The material waspurified via reverse-phase HPLC using a water/acetonitrile gradient andlyophilized to provide a white powder.

r5 conjugate-12 mg obtained (29% isolated yield)

r7 conjugate-22 mg obtained (55% isolated yield)

R7 conjugate-13 mg obtained (33% isolated yield).

Example 17 Penetration of Taxol Conjugated to a Biotinylated Pentamer,Heptamer, and Nonamer of D-Arginine into the Skin of Nude Mice Methods

1. Conjugation of C-2′ Activated Taxol Derivatives to Biotin-LabeledPeptides

Synthesis of C-2′ Derivatives

Taxol (48.7 mg, 57.1 μmol) was dissolved in CH₂Cl₂ (3.0 mL) under anN₂-atmosphere. The solution was cooled to 0° C. A stock solution of thechloroformate of benzyl-(p-hydroxy benzoate) (200 mmol, in 2.0 mLCH₂Cl₂— freshly prepared from benzyl-(p-hydroxy benzoate) anddiphosgene) was added at 0° C. and stirring at that temperature wascontinued for 5 hours, after which the solution was warmed to roomtemperature (FIG. 12). Stirring was continued for additional 10 hours.The solvents were removed in vacuo and the crude material was purifiedby flash chromatography on silica gel (eluent: EtOAc/hexanes 30%-70%)yielding the desired taxol C-2′ carbonate (36.3 mg, 32.8 μmol, 57.4%).

Coupling to Biotin-Labeled Peptides.

A procedure for coupling to biotin-labeled peptides is shown in FIG. 13.The taxol derivative and the biotin labeled peptide (1.2 equivalents)were dissolved in DMF (˜10 μmol/mL DMF) under an N₂-atmosphere. Stocksolutions of diisopropylethylamine (1.2 equivalents in DMF) and DMAP(0.3 equivalents in DMF) were added and stirring at room temperature wascontinued until all starting material was consumed. After 16 hours thesolvent was removed in vacuo. The crude reaction mixture was dissolvedin water and purified by RP-HPLC (eluent: water/MeCN*TFA) yielding theconjugates in the indicated yields:

B-aca-r5-K-taxol: 3.6 mg, 1.32 mmol, 20%.

B-aca-r7-K-taxol: 9.8 mg, 3.01 mmol, 44%.

B-aca-r9-K-taxol: 19.4 mg, 5.1 mmol, 67%.

Unlabeled C-2′ carbamates:

The taxol derivative (12.4 mg, 11.2 μmol) and the unlabeled peptide(27.1 mg, 13.4 μmol) were dissolved in DMF (1.5 mL) under anN₂-atmosphere (FIG. 14). Diisopropylethylamine (1.7 mg, 13.4 μmol) wasadded as a stock solution in DMF, followed by DMAP (0.68 mg, 5.6 μmol)as a stock solution in DMF. Stirring at room temperature was continueduntil all starting material was consumed. After 16 hours the solvent wasremoved in vacuo. The crude material was dissolved in water and purifiedby RP-HPLC (eluent: water/MeCN*TFA) yielding the desired product (16.5mg, 5.9 μmol, 53%).

Other C-2′ Conjugates

The taxol derivative (8.7 mg, 7.85 μmol) was dissolved in EtOAc (2.0mL). Pd/C (10%, 4.0 mg) was added and the reaction flask was purged withH₂ five times (FIG. 15A). Stirring under an atmosphere of hydrogen wascontinued for 7 hours. The Pd/C was filtered and the solvent was removedin vacuo. The crude material (6.7 mg, 6.58 μmol, 84%) obtained in thisway was pure and was used in the next step without further purification.

The free acid taxol derivative (18.0 mg, 17.7 μmol) was dissolved inCH₂Cl₂ (2.0 mL). Dicyclohexylcarbodiimide (4.3 mg, 21.3 μmol) was addedas a stock solution in CH₂Cl₂ (0.1 mL). N-Hydroxysuccinimide (2.0 mg,17.7 μmol) was added as a stock solution in DMF (0.1 mL) (FIG. 15B).Stirring at room temperature was continued for 14 hours. The solvent wasremoved in vacuo and the resultant crude material was purified by flashchromatography on silica gel (eluent: EtOAc/hexanes 40%-80%) yieldingthe desired product (13.6 mg, 12.2 μmol, 69%).

The activated taxol derivative (14.0 mg, 12.6 μmol) and the peptide(30.6 mg, 15.1 μmol) were dissolved in DMF (3.0 mL) under anN₂-atmosphere (FIG. 15C). Diisopropylethylamine (1.94 mg, 15.1 μmol) wasadded as a stock solution in DMF (0.1 mL), followed by DMAP (0.76 mg,6.3 μmol) as a stock solution in DMF 0.1 mL). Stirring at roomtemperature was continued until all the starting material was consumed.After 20 hours the solvent was removed in vacuo. The crude material wasdissolved in water and purified by RP-HPLC (eluent: water/MeCN*TFA)yielding the two depicted taxol conjugates in a ration of 1:6 (carbonatevs carbamate, respectively).

Example 18

This example illustrates a method of linking active agents such asacyclovir to transport moieties. See, FIG. 35.

Acyclovir (1 eq) was dissolved in dry N,N-dimethylformamide under anitrogen atmosphere. Chloroacetic anhydride (1 eq), pyridine (1 eq), andDMAP (0.25 eq) were added subsequently to the reaction with stirring.The reaction was permitted to stir at room temperature for an additional4 hours. The reaction was halted by removal of the solvent under reducedpressure. The residue was dissolved in methylene chloride and washedwith saturated aqueous ammonium chloride followed by saturated aqueousammonium bicarbonate and brine. The organic layer was concentrated invacuo and the residue purified by silica gel chromatography to providethe acyclovir chloroacetyl ester.

The resultant chloroacetyl ester was dissolved in dryN,N-dimethylformamide under a nitrogen atmosphere. To the solution wasadded Hunig's base (1 eq) and AcHN—C-aca-R8-CONH2*8HCl with rapidstirring. The reaction was allowed to proceed until TLC analysisindicated that all of the starting material had been consumed (ca 2hours). The reaction was halted by removal of the solvent under reducedpressure. The residue was purified by RP-HPLC to provide the desiredacyclovir conjugate.

Example 19

This example illustrates a method of linking active agents such asacyclovir to transport moieties. See, FIG. 36.

Disphosgene (0.5 eq) was dissolve in dry methylene chloride and cooledto −10° C. To the solution was added triethylamine (1 eq) as a solutionin methylene chloride. The mixture was stirred for 15 minutes at whichtime acyclovir was added to the reaction as a solution in methylenechloride. The reaction was permitted to stir at room temperature for anadditional 4 hours. The reaction was quenched with saturated aqueousammonium chloride followed by washes of saturated aqueous ammoniumbicarbonate and brine. The organic layer was concentrated in vacuo andthe residue purified by rapid filtration over silica gel to provide theacyclovir chloroformate.

The chloroformate was dissolved in dry methylene chloride under anitrogen atmosphere. Mercaptoethanol (1 eq) was added to the reaction asa solution in dry methylene chloride. The reaction was allowed to stirfor 10 hours under a nitrogen atmosphere. The solution was concentratedunder reduced and placed under high vacuum for 24 hours to removeresidual mercaptoethanol. The resultant mercaptoethyl carbonate was usedwithout further purification.

The carbonate (1 eq) was dissolved DMF/water. To the solution was addedthe activated peptide NPYs-CR*—CONH2*8HCl (1 eq) with rapid stirring. Abright yellow color developed immediately and the reaction was allowedto stir at room temperature for an additional 5 hours. The reaction waspurified directly by RP-HPLC to provide the desired acyclovir conjugate.

Example 20

This example illustrates a method of linking active agents such ascorticoid steroids to transport moieties. See, FIG. 37.

Prednisolone α-chloroester—

To a solution of prednisolone (1.38 mmol), scandium triflate (0.83 mmol)and chloroacetic anhydride (4.14 mmol) in dry THF was addeddimethylaminopyridine (4.14 mmol). The solution turned bright yellowupon addition of dimethylaminopyridine. After 30 minutes the solvent wasevaporated off and the crude material taken up into ethyl acetate (100mL). The ethyl acetate layer was washed with 1.0 N HCl and brine. Theorganic phase was collected, dried (Na₂SO₄) and evaporated to providethe product as a white solid.

H₂N—C(prednisolone)-r8-CONH₂:

A solution of prednisolone α-chloroester (1 equivalent), H₂N—C-r8-CONH₂(1 equivalent) and diisopropylethylamine (1.2 equivalent) indimethylformamide (1 mL) was stirred for 18 hours. The dimethylformamidewas evaporated off. The crude product purified by reverse-phase HPLC (22mm×250 mm C-18 column, a 5-10% CH₃CN/H₂O gradient with 0.1%trifluoroacetic acid, 214 and 254 nm UV detection) and lyophilized toprovide the 9 TFA salt. The material was then subjected to ion exchangechromatography to provide prednisolone conjugate (9 HCl salt) as a tansolid.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1. A method for enhancing delivery of a compound into and across ananimal ocular tissue, the method comprising: administering to the oculartissue a conjugate comprising the compound and a delivery-enhancingtransporter, wherein: i. the compound is attached to thedelivery-enhancing transporter through a linker, and ii. thedelivery-enhancing transporter comprises fewer than 50 subunits andcomprises at least 5 guanidine or amidino moieties, thereby increasingdelivery of the conjugate into the ocular tissue compared to delivery ofthe compound in the absence of the delivery-enhancing transporter. 2.The method of claim 1, wherein delivery of the conjugate into the oculartissue is increased at least two-fold compared to delivery of thecompound in the absence of the delivery-enhancing transporter.
 3. Themethod of claim 1, wherein delivery of the conjugate into the oculartissue is increased at least ten-fold compared to delivery of thecompound in the absence of the delivery-enhancing transporter.
 4. Themethod of claim 1, wherein the ocular tissue is one or more layers ofepithelial or endothelial tissue.
 5. The method of claim 1, wherein theocular tissue is the retina.
 6. The method of claim 1, wherein theocular tissue is the optic nerve.
 7. The method of claim 1, wherein thelinker is a releasable linker.
 8. The method of claim 7, wherein thelinker is stable in a saline solution a pH 7 but is cleaved whentransported into a cell.
 9. The method of claim 1, wherein the subunitsare amino acids.
 10. The method of claim 1, wherein the conjugate has astructure selected from the group consisting of structures 3, 4, or 5,as follows:

wherein: R1 comprises the compound; X is a linkage formed between afunctional group on the biologically active compound and a terminalfunctional group on the linking moiety; Y is a linkage formed from afunctional group on the transport moiety and a functional group on thelinking moiety; A is N or CH; R2 is hydrogen, alkyl, aryl, acyl, orallyl; R3 comprises the delivery'-enhancing transporter; R4 is S, O, NR6or CR7R8; R5 is H, OH, SH or NHR6; R6 is hydrogen, alkyl, aryl, acyl orallyl; k and m are each independently selected from 1 and 2; and n is 1to
 10. 11. The method of claim 10, wherein X is selected from the groupconsisting of —C(O)O—, —C(O)NH—, —OC(O)NH—, —S—S—, C(S)O—, —C(S)NH—,—NHC(O)NH—, —SO₂NH—, —SONH—, phosphate, phosphonate phosphinate, andCR7R8, wherein R7 and R8 are each independently selected from the groupconsisting of H and alkyl.
 12. The method of claim 10, wherein theconjugate comprises structure 3, Y is N, and R2 is methyl, ethyl,propyl, butyl, allyl, benzyl or phenyl.
 13. The method of claim 10,wherein R2 is benzyl; k, m, and n are each 1, and X is —OC(O)—.
 14. Themethod of claim 10, wherein the conjugate comprises structure 4; R4 isS; R5 is NHR6; and R6 is hydrogen, methyl, allyl, butyl or phenyl. 15.The method of claim 10, wherein the conjugate comprises structure 4; R5is NHR6; R6 is hydrogen, methyl, allyl, butyl or phenyl; and k and m areeach
 1. 16. The method of claim 1, wherein the conjugate comprisesstructure 6 as follows:

wherein: R1 comprises the compound; X is a linkage formed between afunctional group on the biologically active compound and a terminalfunctional group on the linking moiety; Y is a linkage formed from afunctional group on the transport moiety and a functional group on thelinking moiety; Ar is an aryl group having the attached radicalsarranged in an ortho or pars configuration, which aryl group can besubstituted or unsubstituted; R3 comprises the delivery-enhancingtransporter; R4 is S, O, NR6 or CR7R8; R5 is H, OH, SH or NHR6; R6 ishydrogen, alkyl, aryl, arylalkyl, acyl or allyl; R7 and R8 areindependently selected from hydrogen or alkyl; and k and m are eachindependently selected from 1 and
 2. 17. The method of claim 16, whereinX is selected from the group consisting of —C(O)O—, —C(O)NH—, —OC(O)NH—,—S—S—, C(S)O—, —C(S)NH—, —NHC(O)NH—, —SO₂NH—, —SONH—, phosphate,phosphonate phosphinate, and CR7R8, wherein R7 and R8 are eachindependently selected from the group consisting of H and alkyl.
 18. Themethod of claim 16, wherein R4 is S; R5 is NHR6; and R6 is hydrogen,methyl, allyl, butyl or phenyl.
 19. The method of claim 1, wherein theconjugate comprises at least two delivery-enhancing transporters. 20.The method of claim 1, wherein the conjugate is administered as an eyedrop. 21-36. (canceled)